An external quality assessment feasibility study; cross laboratory comparison of haemagglutination inhibition assay and microneutralization assay performance for seasonal influenza serology testing: A FLUCOP study.

Introduction: External Quality Assessment (EQA) schemes are designed to provide a snapshot of laboratory proficiency, identifying issues and providing feedback to improve laboratory performance and inter-laboratory agreement in testing. Currently there are no international EQA schemes for seasonal influenza serology testing. Here we present a feasibility study for conducting an EQA scheme for influenza serology methods. Methods: We invited participant laboratories from industry, contract research organizations (CROs), academia and public health institutions who regularly conduct hemagglutination inhibition (HAI) and microneutralization (MN) assays and have an interest in serology standardization. In total 16 laboratories returned data including 19 data sets for HAI assays and 9 data sets for MN assays. Results: Within run analysis demonstrated good laboratory performance for HAI, with intrinsically higher levels of intra-assay variation for MN assays. Between run analysis showed laboratory and strain specific issues, particularly with B strains for HAI, whilst MN testing was consistently good across labs and strains. Inter-laboratory variability was higher for MN assays than HAI, however both assays showed a significant reduction in inter-laboratory variation when a human sera pool is used as a standard for normalization. Discussion: This study has received positive feedback from participants, highlighting the benefit such an EQA scheme would have on improving laboratory performance, reducing inter laboratory variation and raising awareness of both harmonized protocol use and the benefit of biological standards for seasonal influenza serology testing.

Assessment of rat polyclonal antisera's suitability in hemagglutination inhibition assay for influenza surveillance and antigenic mapping.

This paper presents comparative hemagglutination inhibition (HI) assay data obtained using ferret or rat antisera to analyze influenza viruses. The results indicate that rat antisera can be successfully applied both for identification and for antigenic analysis of human influenza A and B viruses. Data gained with rat antisera were comparable to those obtained with ferret antisera. In-depth statistical analysis, based on Confusion Matrix analysis and Receiver Operating Characteristic (ROC) analysis, confirmed good coincidence between ferret antisera-based and rat antisera-based results. Two-dimensional antigenic mapping, based on HI assays using rat and ferret antisera, supported these findings. Both antisera types yielded identical antigenic attributions for the viruses analyzed, and both permitted visualization of contemporary human influenza virus evolutionary trends.

Assessment of hemagglutination activity of porcine deltacoronavirus.

Porcine deltacoronavirus (PDCoV) is an emerging swine enteric coronavirus that causes diarrhea in piglets. However, the biological characteristics of PDCoV are unclear. In this study, the hemagglutination (HA) abilities of two PDCoV strains (CH-01 and HNZK-04) were investigated. Our results showed that PDCoV has the ability to agglutinate rabbit erythrocytes after virion pretreatment with trypsin or neuraminidase. Additionally, the HA assay results showed a significant positive correlation with the infectious viral titer. Our results suggest that assessing the HA activity of PDCoV may be a useful diagnostic method for investigating and surveilling PDCoV infections.

Immunotoxicity assessment of sub-chronic oral administration of acetamiprid in Wistar rats.

CONTEXT: Neonicotinoid insecticides are synthetic analogues of nicotine that acts on the central nervous system of insects by blocking post synaptic acetylcholine receptor. Acetamiprid is one of the widely used neonicotinoid class of insecticide used to control sucking insects like aphids, bees, mosquitoes, on crops. Data on the possible immunotoxic nature of acetamiprid are lacking. OBJECTIVE: The present study was conducted in Wistar rats with the objective of evaluating the immunotoxic potential of acetamiprid administered orally at the dose levels of 27.5, 55 and 110 mg/kg b.w. (equivalent to 5.5, 11 and 22 mg/kg b.w.) for a period of 90 days. MATERIALS AND METHODS: In experiment 1, general toxicity testing including the evaluation of clinical signs, hemato-biochemical changes, response of the lymphocytes towards T and B cell mitogens, macrophage function, gross and histopathology of the lymphoid organs (spleen, thymus, lymph nodes, etc.) were performed. In the second experiment, humoral and cell-mediated responses during immunological challenges were evaluated. RESULTS: Significant decreases were observed in the stimulation index of lymphocyte proliferation to B cell mitogen and in the nitrite production of macrophages of rats treated with 110 mg/kg of acetamiprid. Significant decrease in the lymphoproliferative response towards the B cell mitogen indicated the inability of the B lymphocytes to respond on stimulation that might increase the chances of susceptibility to infections. Acetamiprid also caused 15-28% reduction in nitrite production, an important signal for efficient inflammatory response of macrophages. The functional impairment of macrophages may involve aberrations in the enzymatic degradation of microbes, oxidative burst, generation of free radicals, phagocytosis, release of proinflammatory cytokines and thereby, may hamper host defence causing susceptibility to diseases. No significant changes over hematology, biochemistry, organ weights, histopathology of major immune organs, delayed type hypersensitivity test, response to sRBCs and lymphoproliferation assay for T cell mitogen were observed. CONCLUSION: In conclusion, the results demonstrate for the first time that the subchronic administration of acetamiprid (20% SP-soluble powder) cause significant decreases in the lymphocyte proliferation as well as the macrophage function at the dose level of 110 mg/kg. Considering the chronic population adjusted dose (0.023 mg/kg/day) through dietary exposure for acetamiprid, judicious use of acetamiprid is highly essential. Indiscriminate use of acetamiprid exceeding the doses advised might pose a hazard.

Quantitative assessment of Mycoplasma hemadsorption activity by flow cytometry.

A number of adherent mycoplasmas have developed highly complex polar structures that are involved in diverse aspects of the biology of these microorganisms and play a key role as virulence factors by promoting adhesion to host cells in the first stages of infection. Attachment activity of mycoplasma cells has been traditionally investigated by determining their hemadsorption ability to red blood cells and it is a distinctive trait widely examined when characterizing the different mycoplasma species. Despite the fact that protocols to qualitatively determine the hemadsorption or hemagglutination of mycoplasmas are straightforward, current methods when investigating hemadsorption at the quantitative level are expensive and poorly reproducible. By using flow cytometry, we have developed a procedure to quantify rapidly and accurately the hemadsorption activity of mycoplasmas in the presence of SYBR Green I, a vital fluorochrome that stains nucleic acids, allowing to resolve erythrocyte and mycoplasma cells by their different size and fluorescence. This method is very reproducible and permits the kinetic analysis of the obtained data and a precise hemadsorption quantification based on standard binding parameters such as the dissociation constant K d. The procedure we developed could be easily implemented in a standardized assay to test the hemadsorption activity of the growing number of clinical isolates and mutant strains of different mycoplasma species, providing valuable data about the virulence of these microorganisms.

A study of the transport and immobilisation mechanisms of human red blood cells in a paper-based blood typing device using confocal microscopy.

Recent research on the use of bioactive paper for human blood typing has led to the discovery of a new method for identifying the haemagglutination of red blood cells (RBCs). When a blood sample is introduced onto paper treated with the grouping antibodies, RBCs undergo haemagglutination with the corresponding grouping antibodies, forming agglutinated cell aggregates in the paper. A subsequent washing of the paper with saline buffer could not remove these aggregates from the paper; this phenomenon provides a new method for rapid, visual identification of the antibody-specific haemagglutination reactions and thus the determination of the blood type. This study aims to understand the mechanism of RBC immobilization inside the paper which follows haemagglutination reactions. Confocal microscopy is used to observe the morphology of the free and agglutinated RBCs that are labelled with FITC. Chromatographic elution patterns of both agglutinated and non-agglutinated RBCs are studied to gain insight into the transport behaviour of free RBCs and agglutinated aggregates. This work provides new information about RBC haemagglutination inside the fibre network of paper on a microscopic level, which is important for the future design of paper-based blood typing devices with high sensitivity and assaying speed.

Hemocompatibility assessment of poly(2-dimethylamino ethylmethacrylate) (PDMAEMA)-based polymers.

Poly(2-dimethylamino-ethylmethacrylate) (PDMAEMA), a cationic polymer, has been widely reported as a nonviral carrier. Despite the fact that the cytotoxicity of this polymer has been extensively studied, there is a lack of information about its blood compatibility. Hence, this work evaluates the hemocompatibility of free-form PDMAEMA homopolymers differing in molecular weight (Mw) with or without a poly(ethylene glycol) (PEG) sequence in the form of a palm tree-like structure. Poly(ethylenimine) (PEI) was used as a reference in order to compare its hemoreactivity. Hemagglutination, hemolysis, platelet number, blood coagulation, and the complement systems were assessed in normal human whole blood according to the ISO 10993-4. Results showed that Mw, concentration, and incubation time strongly affected the hemocompatibility of the polymers evaluated. Our in vitro observations highlight that PDMAEMA homopolymers interacted strongly with the surface of the red blood cells but not with the inner structure of the membrane, while PEI behaved in the opposite way. No clear correlation has been evidenced between PDMAEMA-induced hemagglutination, PEI-induced hemagglutination, and hemolysis. Interestingly, if these polyelectrolytes strongly affect the platelets and blood coagulation cascades in a dose dependent way, none of them significantly affects the complement system. Our work reveals new knowledge on the toxicology of 2 families of polycations largely explored for gene delivery and on their mechanisms of cellular and humoral interactions.

Assessment of the efficacy of commercially available and candidate vaccines against a pandemic H1N1 2009 virus.

BACKGROUND: The emergence and global spread of the pandemic H1N1 2009 influenza virus have raised questions regarding the protective effect of available seasonal vaccines and the efficacy of a newly produced matched vaccine. METHODS: Ferrets were immunized with the 2008-2009 formulations of commercially available live attenuated (FluMist; MedImmune) or split-inactivated (Fluviral; GlaxoSmithKline) vaccines, a commercial swine vaccine (FluSure; Pfizer), or a laboratory-produced matched inactivated whole-virus vaccine (A/Mexico/InDRE4487/2009). Adaptive immune responses were monitored, and the animals were challenged with A/Mexico/InDRE4487/2009 after 5 weeks. RESULTS: Only animals that received the swine or matched vaccines developed detectable hemagglutination-inhibiting antibodies against the challenge virus, whereas a T cell response was exclusively detected in animals vaccinated with FluMist. After challenge, all animals had high levels of virus replication in the upper respiratory tract. However, preexisting anti-pandemic H1N1 2009 antibodies resulted in reduced clinical signs and improved survival. Surprisingly, FluMist was associated with a slight increase in mortality and greater lung damage, which correlated with early up-regulation of interleukin-10. CONCLUSIONS: The present study demonstrates that a single dose of matched inactivated vaccine confers partial protection against a pandemic H1N1 2009 virus, and it suggests that a higher dose or prime-boost regimen may be required. The consequences of mismatched immunity to influenza merit further investigation.

A novel and efficient and low-cost methodology for purification of Macrotyloma axillare (Leguminosae) seed lectin.

The N-acetyl-galactosamine specific lectin from Macrotyloma axillare seeds (LMA) was purified by precipitation and ion exchange chromatography. The LMA 0.2 mol L(-1) fraction showed hemagglutinating activity on erythrocytes A1. The results for molecular mass determinations were about 28 kDa. The LMA pH-dependent assays showed best hemagglutinating activity at pH 6.0-8.0; being decreased at acidic/alkaline conditions and by EDTA treatment. LMA is a tetramer at pH 8.2 and a dimer at pH 4.0. Human erythrocytes from ABO system confirmed the A1 specificity for LMA. This new methodology is useful and easy, with low costs, for lectin purification in large amounts.

Assessment of the significance of virulence factors of uropathogenic Escherichia coli in experimental urinary tract infection in mice.

Four Escherichia coli strains, isolated from cystitis patients, belonging to serotype 02:H- and possessing different combinations of urovirulence factors were examined in an experimental pyelonephritis mouse model to assess the relative importance of virulence factors in causation of urinary tract infections (UTI). The results suggest not only that the each virulence factor has a role in causation of UTI but also that the presence of P fimbriae and production of hemolysin significantly reduced the LD50 and ID50 of the strains in the mouse model. The results also demonstrate that the presence of additional virulence factors acts in an additive or synergetic fashion enhancing the cumulative impact of the strain.

BCSH-NIBSC anti-D reference reagent for antiglobulin tests: the in-house assessment of red cell washing centrifuges and of operator variability in the detection of weak, macroscopic agglutination. British Committee for Standards in Haematology. National Institute for Biological Standards and Control.

A batch of an anti-D preparation, reference 91/608, has been prepared for the preparation of red cells weakly sensitized with IgG that can reveal inhibition of the antiglobulin test by one volume of human serum, diluted 1:1000. The preparation provides an objective assessment of red cell washer efficacy and the confidential, in-house assessment of operator variability in detecting weak but definite macroscopic agglutination by blind, replicate tests. Red cell washer efficacy and poor operator reading procedures causing disruption of weak agglutination are two major causes of false-negative antiglobulin tests; neither are adequately detected by the common quality-control procedure of adding strongly IgG-sensitized red cells ('Coombs control cells') to apparently negative antiglobulin tests. However, weakly IgG-sensitized red cells do offer a valuable control function that can detect some degree of cell washer inefficiency and reading errors although such cells are not a substitute for the more sensitive replicate testing. Test protocols are provided to assess the efficacy of cell washing machines and operator skills in the detection of weak but definite macroscopic agglutination.

2-Mercapto-ethanol enhancement of agglutination reaction--a possible in vitro serological correlate for assessment of functional immunity in simian malaria.

Conventional indirect haemagglutination test was performed in rhesus monkey sera (collected from Plasmodium knowlesi infected animals) with and without prior treatment of sera with 2-mercapto-ethanol (2-ME). Surprisingly, many sera samples showed significant enhancement of final titre with 2-ME. The 2-ME enhancement effect was more pronounced in the sera of hyperimmune monkeys on further injection of antigen or parasites. It was also noticeable in the sera during primary drug-suppressed P. knowlesi infection and appeared to have a bearing on the immune status of the animals to rechallenge. The use of a soluble antigen prepared from P. knowlesi infected erythrocytes was found to be essential in IHA test to demonstrate the 2-ME enhancement effect. Antigen prepared from freed parasites (commonly used) failed to show a similar effect in IHA. The possible role of certain T-lymphocyte products - antigen binding, non-agglutinating, 2-ME sensitive molecules - in malarial immunology has been proposed.