A review of analytical parameters in 'rapid' liquid chromatographic methods for bioanalysis: Can we do better?

Rapid bioanalysis is beneficial to many applications. However, how 'rapid' a method is, or could be, is often an unanswered question. In this statistical review, the authors have assessed multiple pre-analytical (i.e. sample preparation), and analytical method parameters specifically for liquid chromatography to assist researchers in developing and validating 'rapid' bioanalytical methods. We restricted the search to urine and plasma matrices only. Data were extracted from over 2,000 recent studies and evaluated to assess how these parameters affected the 'on-instrument' analysis time. In addition to methods using ultra-violet (UV) detection, there were a large number of mass spectrometric (MS) methods, allowing additional review of the differences between high- and low-resolution MS on analysis time. We observed that most (N = 922, 70 %) methods used 5 or 10 cm columns, and that whilst uptake of ultra-high performance (U)HPLC columns was good, the use of sub-5 cm columns and/or flow rates in excess of 1 mL/min was incredibly rare (N = 25, 3 %). The detector of choice for quantitative (U)HPLC-MS remains the triple quadrupole, although a number of groups report the use of high-resolution MS for such methods.

The application of FAIMS gas analysis in medical diagnostics.

There is an ever increasing need to develop new tools to aid in the diagnosis and monitoring of human diseases. Such tools will ultimately reduce the cost of healthcare by identifying disease states more quickly and cheaply than current practices. One method showing promise is the analysis of gas-phase biomarkers from human breath, urine, sweat and stool that reflect bodily metabolism. Analysis of these volatiles by GC MS requires specialised infra-structure and staff, making it unsuitable for a clinical setting. Point of care sensor based technologies such as eNoses often suffer from stability and sensitivity issues. Field-Asymmetric Ion Mobility Spectrometry (FAIMS) has potential to fulfil this clinical need. In this paper we review the medical need, the technology, sampling methods and medical evidence thus far. We conclude with reflecting on future developmental steps necessary to bring the device into medical practice.

Hydroxyapatite ceramic-coated femoral components in young patients followed up for 17 to 25 years: an update of a previous report.

This study reports the results of 38 total hip arthroplasties (THAs) in 33 patients aged < 50 years, using the JRI Furlong hydroxyapatite ceramic (HAC)-coated femoral component. This represents an update of previous reports of the same cohort at ten and 16 years, which were reported in 2004 and 2009, respectively. We describe the survival, radiological and functional outcomes at a mean follow-up of 21 years (17 to 25). Of the surviving 34 THAs, one underwent femoral revision for peri-prosthetic fracture after 21 years, and one patient (one hip) was lost to follow-up. Using aseptic loosening as the end-point, 12 hips (31.5%) needed acetabular revision but none needed femoral revision, demonstrating 100% survival (95% confidence interval 89 to 100). In young patients with high demands, the Furlong HAC-coated femoral component gives excellent long-term results.

Extraction methods for the removal of phospholipids and other endogenous material from a biological fluid.

BACKGROUND: A comparison of three different sample preparation techniques for the analysis of plasma samples has been investigated to highlight the effect that these approaches have on the removal of endogenous material. The three techniques under investigation are: SPE, support assisted liquid-liquid extraction and nonspecific solvent-based protein precipitation. RESULTS: Comparisons are made on the practicalities of each approach and to allow a semiquantitative assessment between the effectiveness of these different techniques the relative amounts of phospholipids present within the sample are analyzed. Total ion chromatograms are also obtained to further study the effects of different extraction techniques in the removal of endogenous components from a biological matrix. Both of these approaches provide a very coarse measure of the cleanliness of the extracts and demonstrate that support assisted liquid-liquid extraction and an optimized SPE approach remove a greater amount of endogenous material. CONCLUSION: This study highlights the importance of sample preparation in removing endogenous material, which may have a detrimental effect on the performance of a bioanalytical assay.

Intense star formation within resolved compact regions in a galaxy at z = 2.3.

Massive galaxies in the early Universe have been shown to be forming stars at surprisingly high rates. Prominent examples are dust-obscured galaxies which are luminous when observed at sub-millimetre wavelengths and which may be forming stars at a rate of 1,000 solar masses (M(middle dot in circle)) per year. These intense bursts of star formation are believed to be driven by mergers between gas-rich galaxies. Probing the properties of individual star-forming regions within these galaxies, however, is beyond the spatial resolution and sensitivity of even the largest telescopes at present. Here we report observations of the sub-millimetre galaxy SMMJ2135-0102 at redshift z = 2.3259, which has been gravitationally magnified by a factor of 32 by a massive foreground galaxy cluster lens. This magnification, when combined with high-resolution sub-millimetre imaging, resolves the star-forming regions at a linear scale of only 100 parsecs. We find that the luminosity densities of these star-forming regions are comparable to the dense cores of giant molecular clouds in the local Universe, but they are about a hundred times larger and 10(7) times more luminous. Although vigorously star-forming, the underlying physics of the star-formation processes at z approximately 2 appears to be similar to that seen in local galaxies, although the energetics are unlike anything found in the present-day Universe.

Hydroxyapatite-ceramic-coated femoral components in young patients followed-up for 16 to 19 years: an update of a previous report.

In 2004 we described the ten-year prospective results of 38 total hip replacements using the Furlong hydroxyapatite-ceramic-coated femoral component in 35 patients < 50 years old. We have now reviewed the surviving 35 arthroplasties in 33 patients at a mean of 16 years (10.3 to 19.9). The mean age of the surviving patients at the time of operation was 41.3 years (26.0 to 49.0). Of these, eight have undergone revision of their acetabular component for aseptic loosening. None of the femoral components has had revision for aseptic loosening giving a survival rate of 100% at 16 years (95% confidence interval 89% to 100%). The Furlong hydroxyapatite-ceramic-coated femoral component gives excellent long-term survival in young and active patients.

Temperature as a variable in liquid chromatography: development and application of a model for the separation of model drugs using water as the eluent.

The use of high temperatures in liquid chromatography allows for the use of a purely aqueous mobile phase. At elevated temperatures water possesses many of the characteristics of organic solvents in terms of eluotropic strength, as well as having a lower viscosity. A model is developed, based on data obtained using a range of model drugs, which demonstrates the relationship between temperature, flow and pressure. Experimental data from different column types, at temperatures from 40 degrees C to 180 degrees C, is presented which matches well with the predicted data from the model.

Hydroxyapatite-ceramic-coated femoral stems in revision hip surgery.

We describe the clinical and radiological results of 120 consecutive revision hip replacements in 107 patients, using the JRI Furlong hydroxyapatite-ceramic-coated femoral component. The mean age of the patients at operation was 71 years (36 to 92) and the mean length of follow-up 8.0 years (5.0 to 12.4). We included patients on whom previous revision hip surgery had taken place. The patients were independently reviewed and scored using the Harris hip score, the Western Ontario and McMaster Universities osteoarthritis index (WOMAC) and the Charnley modification of the Merle d'Aubigné and Postel score. Radiographs were assessed by three reviewers for the formation of new bone, osteolysis, osseointegration and radiolucent lines in each Gruen zone. The mean Harris hip score was 85.8 (42 to 100) at the latest post-operative review. The mean WOMAC and Merle d'Aubigné and Postel scores were 34.5 and 14.8, respectively. The mean visual analogue score for pain (possible range 0 to 10) was 1.2 overall, but 0.5 specifically for mid-thigh pain. There were no revisions of the femoral component for aseptic loosening. There were four re-revisions, three for infection and one for recurrent dislocation. Radiological review of all the femoral components, including the four re-revisions showed stable bony ingrowth and no new radiolucent lines in any zone. Using revision or impending revision for aseptic loosening as an end-point, the cumulative survival of the femoral component at ten years was 100% (95% confidence interval 94 to 100). We present excellent medium- to long-term clinical, radiological and survivorship results with the fully hydroxyapatite-ceramic-coated femoral component in revision hip surgery.

The lateral cerebellum and visuomotor control.

The lateral cerebellum receives an abundance of visual input providing the link between visual and motor control centers. In this review we discuss experiments designed to increase our understanding of how visual inputs to the cerebellum are arranged in relation to the zonal organization of the cerebellar cortex, and how visual inputs are utilized to assist in the regulation of a visually guided movement. On the basis of anatomical and physiological characteristics our findings indicate that the medial-most folium in crus I of the cat lateral cerebellum can be subdivided into at least three functionally distinct zones; from lateral to medial along the length of the folium these correspond to zones D(1), lateral C(3) and C(2). Each zone displays clear differences in olivo-cortico-nuclear connectivity and in the anesthetized animal zones D(1) and C(2) both receive powerful visual inputs relayed via the climbing fiber system. Complementary experiments in awake behaving cats found that Purkinje cells located in the D(1) and D(2) zones of crus I exhibit changes in simple spike discharge time locked to target motion during a visually guided reaching task. These changes were unaffected by temporary visual denial of the target, raising the possibility that internally generated feedforward visuomotor control mechanisms are operating, in which a predictive model of the target's motion has been constructed by the CNS.

Hydroxyapatite ceramic-coated femoral stems in young patients. A prospective ten-year study.

We describe the clinical and radiological results of 38 total hip replacements (THR) using the JRI Furlong hydroxyapatite-ceramic (HAC)-coated femoral component in patients younger than 50 years. The mean age at the time of operation was 42 years (22 to 49) and the mean length of follow-up was ten years (63 to 170 months). All patients receiving a Furlong HAC THR were entered into the study regardless of the primary pathology including patients who had undergone previous hip surgery. The mean Harris hip score improved from 44 before operation to 92 at the last post-operative review. After 12 years the cumulative surivival for the stem was 100% (95% confidence interval 89 to 100). No femoral component was revised. Our results show that the Furlong HAC implant gives excellent long-term results in young patients with high demands.

Outcome following a second hip fracture.

Hip fracture has a significant impact on medical resources and patient morbidity and mortality. Few patients admitted with a hip fracture are considered for prophylactic measures aimed at the reduction of further fracture risk. Currently, 10-13% of patients will later sustain a second hip fracture. In considering the possible role of prophylactic measures in the prevention of second hip fracture, we investigated whether second hip fracture has a significant further impact on patients' mobility and social independence. We also sought to identify a pattern to second hip fracture that may allow targeting of those patients who are most likely to benefit from currently available prophylactic measures. We undertook a retrospective review of 49 consecutive patients admitted with a second hip fracture between August 1999 and September 2000. Fewer patients maintained their ability to walk independently after the second fracture than did so after the first (53 and 91% respectively, P<0.0005). Following second hip fracture, patients' level of mobility determined their future social independence. Older patients and those with a history of multiple falls had a shorter time interval between fractures. Second hip fracture has a significant further impact on patients' mobility and social independence. Younger patients and those without a history of multiple falls are most likely to benefit from medical prophylaxis.

Cell therapy attenuates deleterious ventricular remodeling and improves cardiac performance after myocardial infarction.

BACKGROUND: Myocardial infarction (MI) promotes deleterious remodeling of the myocardium, resulting in ventricular dilation and pump dysfunction. We examined whether supplementing infarcted myocardium with skeletal myoblasts would (1) result in viable myoblast implants, (2) attenuate deleterious remodeling, and (3) enhance in vivo and ex vivo contractile performance. METHODS AND RESULTS: Experimental MI was induced by 1-hour coronary ligation followed by reperfusion in adult male Lewis rats. One week after MI, 10(6) myoblasts were injected directly into the infarct region. Three groups of animals were studied at 3 and 6 weeks after cell therapy: noninfarcted control (control), MI plus sham injection (MI), and MI plus cell injection (MI+cell). In vivo cardiac function was assessed by maximum exercise capacity testing and ex vivo function was determined by pressure-volume curves obtained from isolated, red cell-perfused, balloon-in-left ventricle (LV) hearts. MI and MI+cell hearts had indistinguishable infarct sizes of approximately 30% of the LV. At 3 and 6 weeks after cell therapy, 92% (13 of 14) of MI+cell hearts showed evidence of myoblast graft survival. MI+cell hearts exhibited attenuation of global ventricular dilation and reduced septum-to-free wall diameter compared with MI hearts not receiving cell therapy. Furthermore, cell therapy improved both post-MI in vivo exercise capacity and ex vivo LV systolic pressures. CONCLUSIONS: Implanted skeletal myoblasts form viable grafts in infarcted myocardium, resulting in enhanced post-MI exercise capacity and contractile function and attenuated ventricular dilation. These data illustrate that syngeneic myoblast implantation after MI improves both in vivo and ex vivo indexes of global ventricular dysfunction and deleterious remodeling and suggests that cellular implantation may be beneficial after MI.

A specific structural alteration in the heparan sulphate of human glomerular basement membrane in diabetes.

AIMS/HYPOTHESIS: Heparan sulphate proteoglycan is an important component of the glomerular anionic filtration barrier and its reduced amount in diabetes contributes to glomerular dysfunction. The objective of this study was to determine if there is also an alteration in the sulphation pattern of the diabetic heparan sulphate chains. METHODS: The heparan sulphate in the glomerular basement membrane/mesangial matrix from human diabetic and nondiabetic kidneys obtained at autopsy was fragmented by a hydrazine/nitrous acid procedure and after radiolabelling with NaB[3H]4, the disaccharide products were chromatographically resolved and quantified. RESULTS: Six sulphated disaccharides were identified in both the diabetic and nondiabetic samples and the molar distribution of these was similar, with the notable exception of the iduronic acid-2-O-sulphatectl--> 4glucosamine-3-O-sulphate species which occurred in the diabetic glomeruli in less than half the amount as in the nondiabetic samples (9.0% compared to 18.7% of total sulphated disaccharides, p < 0.005). CONCLUSION/INTERPRETATION: 3-O-sulphated glucosamine is a rare constituent of heparan sulphate occurring usually in a glucuronic acidbeta1--> 4glucosamine-3-O-sulphate(+/- 6-O-sulphate) sequence within the antithrombin-binding domain. In the glomerular basement membrane where the 3-O-sulphated glucosamine is present in substantial amounts, however, it occurs exclusively in an iduronic acid-containing sequence. It is likely that the recently discovered 3-O-sulphotransferase variant which specifically acts on the iduronic acidalpha1--> 4glucosamine sequence is decreased in human diabetes and moreover that this unusual disaccharide could be a component of a specific heparan sulphate domain which interacts with bioactive proteins.

Surface temperature-field imaging with laser-induced thermographic phosphorescence.

A technique to remotely image temperature distributions of heated metallic surfaces is extended to higher temperatures. It uses a Dy(+3):YAG thermographic phosphor (TP) bonded to the surface and excited by radiation at 355 nm. Digital images of the emission from two excited states were recorded and divided by each other to correct by normalization for illumination and coating nonuniformities. Results show that the TP can survive heating and cooling cycles to 1400 K and that emitting states achieve thermodynamic equilibrium before radiating. Temperatures in the range of 300-1300 K were determined by normalization of pairs of emission images with a single calibration constant. Uncertainties of +/-7-13% at a spatial resolution of 20 microm and +/-0.7-4% at a resolution of 500 microm were achieved.

Human anti-porcine T cell response: blocking with anti-class I antibody leads to hyporesponsiveness and a switch in cytokine production.

Intervention in the molecular interactions that lead to an immune response is possible at various stages of Ag recognition and T cell activation. Perturbation of the interaction of the TCR with the MHC/peptide ligand complex is one approach that has shown promise for autoimmunity and graft rejection in blocking T cell-activated responses. In this study, we investigated the effect of altering the target MHC class I molecule by blocking with Abs. We established a system that analyzed the human T cell response against MHC class I+/class II- porcine stimulatory cell targets. The primary human response against porcine smooth muscle cells was CD8+ T cell dependent. In the presence of F(ab')2 fragments of the MHC class I-reactive Ab, PT-85, the proliferative response was inhibited and production of IL-2 and IFN-gamma was blocked. Moreover, in a secondary response, proliferation was reduced and type 1 cytokine levels were inhibited. In contrast, levels of IL-10 and IL-4 were sustained or slightly increased. These findings indicate that Ab against MHC class I blocked the recognition of porcine cells by the human CD8+ T cells and altered the cytokine secretion profile. Thus, a single treatment with PT-85 F(ab')2 directed against the MHC class I molecule provides an attractive approach to the induction of T cell tolerance that may provide long-term graft survival in porcine-to-human cell transplantation.

Xenogeneic transplantation of porcine hepatocytes into the CCl4 cirrhotic rat model.

Liver support using extracorporeal devices and hepatocyte transplantation has received renewed interest for the management of acute and chronic liver failure. The aim of this study was to determine whether xenogeneic porcine hepatocytes could integrate into the liver parenchyma of cirrhotic Lewis rats when administered by an intrasplenic route. Cirrhosis was induced by carbon tetrachloride (CCl4) inhalation and confirmed histologically. Freshly isolated porcine hepatocytes were infused directly into the splenic pulp at laparotomy over a 5-15-min interval. Using (111)In-labeled hepatocytes, the degree of localization of porcine hepatocytes to the spleen and liver was found to be greater than 60% in both control and cirrhotic rats. Integration of porcine hepatocytes into the rat liver parenchyma was determined by immunohistochemical staining for porcine albumin in rat liver sections. Further confirmation was provided by in situ hybridization using a porcine-specific probe that binds to a distinct repetitive element (PRE) in porcine DNA. Evidence of integrated porcine hepatocytes was seen for over 50 days in animals under cyclosporine immunosuppression. These data demonstrate the integration of xenogeneic porcine hepatocytes into the liver of the cirrhotic rat and their ability to produce porcine albumin for up to 50 days.

Xenogeneic cell therapy: current progress and future developments in porcine cell transplantation.

The multitude of distinct cell types present in mature and developing tissues display unique physiologic characteristics. Cellular therapy is a novel technology with the promise of utilizing this diversity to treat a wide range of human degenerative diseases. Intractable diseases, disorders, and injuries are characterized by cell death or aberrant cellular function. Cell transplantation can replace diseased or lost tissue to provide restorative therapy for these conditions. The limited use of cell transplants as a basis for current therapy can, in part, be attributed to the lack of available human cells suitable for transplantation. This has prevented further realization of the promise of cell transplantation as a platform technology. Accordingly, cell-based therapies such as blood transfusions, for which the cells are readily available, are a standard part of current medical practice. Despite numerous attempts to expand primary human cells in tissue culture, current technological limitations of this approach in regard to proliferative capacity and maintenance of the differentiated phenotype has prevented their use for transplantation. Further, use of human stem cells for the derivation of specific cell types for transplantation is an area of future application with great potential, but hurdles remain in regard to deriving and sufficiently expanding these multipotential cells. Thus, it appears that primary cells are at present a superior source for transplantation. This review focuses on pigs as a source of a variety of primary cells to advance cell therapy to the clinic and implement achievement of its full potential. We outline the advantages and disadvantages of xenogeneic cell therapy while underscoring the utility of transplantable porcine cells for the treatment of human disease.

Analysis of polymorphism in porcine MHC class I genes: alterations in signals recognized by human cytotoxic lymphocytes.

Elucidation of the mechanism of the immune response against transplanted porcine tissue is critical for the success of xenografting in humans. Both human T cells and NK cells recognize MHC Ags, and human receptors may bind to MHC Ags across species barriers. Molecular characterization of porcine MHC class I clones from two MHC class I loci (P1 and P14) obtained from homozygous inbred miniature swine of three haplotypes (aa, cc, and dd), revealed extensive conservation between loci, suggesting that the genes were products of duplication from a common ancestral sequence. The level of homology between loci was similar to that between the haplotypes at each locus, suggesting that intergenic exchange had limited divergence of these genes. Comparison of the alleles indicated that the polymorphism occurred in the alpha-1 and alpha-2 domains of the class I heavy chain, while the alpha-3 domain was highly conserved among the six genes analyzed. Amino acids in the alpha-2 and alpha-3 domains responsible for the binding of human CD8 to MHC class I were largely conserved in the porcine genes, but several critical residues were altered. Comparison of sequences recognized by human NK cell inhibitory receptors revealed that the residues critical for recognition by these receptors were altered in the porcine genes; thus, the porcine class I molecules would be unable to inhibit lysis by human NK clones characterized to date. This finding provides a likely explanation for the susceptibility of porcine cells to cytolysis by human NK cells.