5-lipoxygenase-dependent biosynthesis of novel 20:4 n-3 metabolites with anti-inflammatory activity.

5-lipoxygenase (5-LO) catalyzes the conversion of arachidonic acid (AA) into pro-inflammatory leukotrienes. N-3 PUFA like eicosapentaenoic acid are subject to a similar metabolism and are precursors of pro-resolving mediators. Stearidonic acid (18:4 n-3, SDA) is a plant source of n-3 PUFA that is elongated to 20:4 n-3, an analogue of AA. However, no 5-LO metabolites of 20:4 n-3 have been reported. In this study, control and 5-LO-expressing HEK293 cells were stimulated in the presence of 20:4 n-3. Metabolites were characterized by LC-MS/MS and their anti-inflammatory properties assessed using AA-induced autocrine neutrophil stimulation and leukotriene B4-mediated chemotaxis. 8­hydroxy­9,11,14,17-eicosatetraenoic acid (Δ17-8-HETE) and 8,15-dihydroxy-9,11,13,17-eicosatetraenoic acid (Δ17-8,15-diHETE) were identified as novel metabolites. Δ17-8,15-diHETE production was inhibited by the leukotriene A4 hydrolase inhibitor SC 57461A. Autocrine neutrophil leukotriene stimulation and neutrophil chemotaxis, both BLT1-dependent processes, were inhibited by Δ17-8,15-diHETE at low nM concentrations. These data support an anti-inflammatory role for Δ17-8,15-diHETE, a novel 5-LO product.

Current laboratory practices in flow cytometry for the enumeration of CD 4(+) T-lymphocyte subsets.

BACKGROUND: CD4(+) T-lymphocyte subset enumeration is routinely used for monitoring HIV disease progression, with approximately 300,000 tests performed annually in the UK alone. Technical variables can impact upon any laboratory test and therefore the final result obtained. Here, we report the findings of a survey questionnaire issued to 1,587 clinical flow cytometry laboratories to: (a) determine if the UK NEQAS for Leucocyte Immunophenotyping (UK NEQAS LI) lymphocyte subset external quality assessment (EQA) programme was suitable for current laboratory needs and practices; and (b) assess the impact of these responses on clinical practice where CD4(+) T-lymphocyte subsets analysis is undertaken. The survey covered areas not traditionally examined by EQA such as: staffing numbers, flow cytometer age and service intervals, plus six test specific sections covering: leukaemia immunophenotyping, CD4(+) T-lymphocyte subsets analysis (reported here), CD34(+) stem cell testing, low level leucocyte enumeration, minimal residual disease testing and PNH testing. RESULTS: The responses revealed major methodological variations between centres undertaking CD4(+) T-lymphocyte subset analysis. Significant differences existed in basic laboratory practices such as: normal range derivation; pipetting techniques; instrument maintenance and units of reporting, all of which results in non-adherence to international guidelines. DISCUSSION: Despite the availability of international guidelines our survey highlighted a lack of concordance amongst laboratory techniques. Such variation could adversely impact on patient care and clinical trial data. Therefore, it is recommended centres undertaking flow cytometric CD4(+) T-lymphocyte subsets analysis urgently review their methodologies and normal ranges to ensure they are fit for purpose and meet current international guidelines.

Specialist integrated haematological malignancy diagnostic services: an Activity Based Cost (ABC) analysis of a networked laboratory service model.

AIMS: Specialist Integrated Haematological Malignancy Diagnostic Services (SIHMDS) were introduced as a standard of care within the UK National Health Service to reduce diagnostic error and improve clinical outcomes. Two broad models of service delivery have become established: 'co-located' services operating from a single-site and 'networked' services, with geographically separated laboratories linked by common management and information systems. Detailed systematic cost analysis has never been published on any established SIHMDS model. METHODS: We used Activity Based Costing (ABC) to construct a cost model for our regional 'networked' SIHMDS covering a two-million population based on activity in 2011. RESULTS: Overall estimated annual running costs were £1 056 260 per annum (£733 400 excluding consultant costs), with individual running costs for diagnosis, staging, disease monitoring and end of treatment assessment components of £723 138, £55 302, £184 152 and £94 134 per annum, respectively. The cost distribution by department was 28.5% for haematology, 29.5% for histopathology and 42% for genetics laboratories. Costs of the diagnostic pathways varied considerably; pathways for myelodysplastic syndromes and lymphoma were the most expensive and the pathways for essential thrombocythaemia and polycythaemia vera being the least. CONCLUSIONS: ABC analysis enables estimation of running costs of a SIHMDS model comprised of 'networked' laboratories. Similar cost analyses for other SIHMDS models covering varying populations are warranted to optimise quality and cost-effectiveness in delivery of modern haemato-oncology diagnostic services in the UK as well as internationally.

99mTc-NC100668, an agent for imaging venous thromboembolism: The effect of anticoagulant or thrombolytic therapy on the uptake and retention of radioactivity in blood clots in vivo.

BACKGROUND: The purpose of this study was to evaluate the uptake of (99m)Tc-NC100668 into blood clots and elucidate the potential for medications commonly used to treat thromboembolism to interfere with the uptake and retention of (99m)Tc-NC100668. METHODS: (99m)Tc-NC100668 in vivo uptake and retention in a range of blood clot of various ages (up to 4 h. old) and in the presence of anticoagulants or thrombolytic therapies was measured in a rat model of deep vein thrombosis. RESULTS: (99m)Tc-NC100668 was rapidly absorbed into and retained by blood clots and was not significantly affected by the presence of unfractionated or low molecular weight heparin or thrombin inhibitor. Tissue plasminogen activator reduced the uptake of (99m)Tc-NC100668 into blood clot by a factor of 3 when adjusted to allow for changes in the weight of the blood clot. CONCLUSIONS: This study has demonstrated that the uptake and retention of (99m)Tc-NC100668 into blood clots in the rat model of deep vein thrombosis is rapid and maintained over at least a 4 h. post-injection period. It has been shown that (99m)Tc-NC100668 is retained in blood clots even in the presence of therapeutic doses of those anticoagulant and thrombolytic therapies typically used to treat pulmonary embolism and venous thrombosis.

(99m)Tc-NC100668, a new tracer for imaging venous thromboemboli: pre-clinical biodistribution and incorporation into plasma clots in vivo and in vitro.

PURPOSE: (99m)Tc-NC100668 is a new radiotracer being developed to aid the diagnosis of thromboembolism. The structure of NC100668 is similar to a region of human alpha(2)-antiplasmin, which is a substrate for factor XIIIa (FXIIIa). The purpose of this study was to confirm the uptake of (99m)Tc-NC100668 into forming plasma clot and to establish the biodistribution of (99m)Tc-NC100668 in Wistar rats. METHODS: The in vitro plasma clot uptake of (99m)Tc-NC100668 and other compounds with known affinities to FXIIIa was measured using a plasma clot assay. The biodistribution and blood clot uptake of radioactivity of (99m)Tc-NC100668 in normal Wistar rats and those bearing experimentally induced deep vein thrombi were investigated. RESULTS: The in vitro uptake of (99m)Tc-NC100668 was greater than that for [(14)C]dansyl cadaverine, a known substrate of FXIIIa in the plasma clot assay. The biodistribution of (99m)Tc-NC100668 in male and female Wistar rats up to 24 h p.i. showed that radioactivity was rapidly excreted, predominantly into the urine, with very little background tissue retention. In vivo the uptake and retention of (99m)Tc-NC100668 into the blood clot was greater than could be accounted for by non-specific accumulation of the radiotracer within the blood clot. CONCLUSION: (99m)Tc-NC100668 was retained by plasma clots in vitro and blood clots in vivo. No significant tissue retention which could interfere with the ability to image thrombi in vivo was observed. This evidence suggests that (99m)Tc-NC100668 might be useful in the detection of thromboembolism.

The in vivo and in vitro metabolic profile of 99mTc-NC100668, a new tracer for imaging venous thromboembolism: identification and biodistribution of the principal radiolabeled metabolite.

(99m)Tc-NC100668 [Acetyl-Asn-Gln-Glu-Gln-Val-Ser-Pro-Tyr(3-iodo)-Thr-Leu-Leu-Lys-Gly-NC100194] is a radiopharmaceutical imaging agent being developed to aid the diagnosis of thromboembolism. The stability profile of (99m)Tc-NC100668 was investigated by high-performance liquid chromatography (HPLC) after in vitro exposure to blood and plasma obtained from rat and human, as well as to urine and bile obtained from rat. The metabolic profile of (99m)Tc-NC100668 exposed to human and rat hepatic S9 (a liver homogenate-rich cytochrome P450) was also studied. The profile of (99m)Tc-labeled species in plasma, urine, and bile was investigated following i.v. administration of (99m)Tc-NC100668 to rat. The major species observed in vitro and in vivo consisted of the (99m)Tc-chelator (NC100194) [N,N-Bis(N-(1,1-dimethyl-2-(hydroxylimino-)propyl)aminoethyl)aminoethylamine] attached to the C-terminal amino acid residue and referred to as (99m)Tc-complex of Gly-NC100194. The identity of the major metabolite was confirmed by cochromatography with an authentic standard and the genuine metabolite using a second HPLC method. The minor metabolites were sodium pertechnetate ((99m)Tc) and (99m)Tc-NC100194. In addition, a small number of other species were transiently observed in vitro; they were not investigated further. The biodistribution of the major metabolite was studied in male Wistar rats. The affinity of the major metabolite toward plasma clot was established using a plasma clot-forming assay. A minor uptake of (99m)Tc-complex of Gly-NC100194 in the plasma clot and a rapid removal from the body were noted. In conclusion, the metabolites of (99m)Tc-NC100668 are not anticipated to have a negative impact on the ability of the test substance to image blood clots.

Androgen excess fetal programming of female reproduction: a developmental aetiology for polycystic ovary syndrome?

The aetiology of polycystic ovary syndrome (PCOS) remains unknown. This familial syndrome is prevalent among reproductive-aged women and its inheritance indicates a dominant regulatory gene with incomplete penetrance. However, promising candidate genes have proven unreliable as markers for the PCOS phenotype. This lack of genetic linkage may represent both extreme heterogeneity of PCOS and difficulty in establishing a universally accepted PCOS diagnosis. Nevertheless, hyperandrogenism is one of the most consistently expressed PCOS traits. Animal models that mimic fetal androgen excess may thus provide unique insight into the origins of the PCOS syndrome. Many female mammals exposed to androgen excess in utero or during early post-natal life typically show masculinized and defeminized behaviour, ovulatory dysfunction and virilized genitalia, although behavioural and ovulatory dysfunction can coexist without virilized genitalia based upon the timing of androgen excess. One animal model shows particular relevance to PCOS: the prenatally androgenized female rhesus monkey. Females exposed to androgen excess early in gestation exhibit hyperandrogenism, oligomenorrhoea and enlarged, polyfollicular ovaries, in addition to LH hypersecretion, impaired embryo development, insulin resistance accompanying abdominal obesity, impaired insulin response to glucose and hyperlipidaemia. Female monkeys exposed to androgen excess late in gestation mimic these programmed changes, except for LH and insulin secretion defects. In utero androgen excess may thus variably perturb multiple organ system programming and thereby provide a single, fetal origin for a heterogeneous adult syndrome.

Diagnosis of plasma cell leukaemia: findings of the UK NEQAS for Leucocyte Immunophenotyping scheme.

The diagnosis of plasma cell leukaemia, a rare disorder with an aggressive clinical course and poor prognosis, is not always straightforward and may be dependent on the results of immunophenotyping. Samples from two cases of plasma cell leukaemia have been issued by the UK NEQAS for Leucocyte Immunophenotyping Scheme during the last 5 years and on each occasion a significant number of laboratories failed to make the correct diagnosis. The details of the two samples issued and the results of both surveys are presented. The data highlights the need to adhere to guidelines for immunophenotyping, with respect to using the correct antibody panels, the importance of data interpretation in conjunction with morphological appearance as well as the need to participate in external quality assurance schemes.

Impact of standardization on clinical cell analysis by flow cytometry.

The evolution of flow cytometry from a research tool to a pivotal technology for clinical diagnostic purposes has required significant efforts to standardize methods. The great advantage of flow cytometry is that it's applications are highly amenable to standardization. Here, we review the efforts that have been made for flow cytometric applications in four major fields of clinical cell analysis: CD4+ T-cell enumeration, CD34+ hematopoietic stem and progenitor cell enumeration, screening for the HLA-B27 antigen and leukemia/lymphoma immunophenotyping. These standardization efforts have been parallelled by the establishment of external quality assessment (EQA) schemes in many countries worldwide. The goal of these EQA exercises has been primarily educa-tional, but their results will increasingly serve as a basis for laboratory accreditation. This important development requires that the EQA schemes, in particular the quality of the distributed samples and the procedures for evaluating the results, meet the highest standards.

Validation of the single-platform ISHAGE method for CD34(+) hematopoietic stem and progenitor cell enumeration in an international multicenter study.

BACKGROUND: Flow cytometric enumeration of CD34+ hematopoietic sterm and progenitor cells (HPC) is the reference point for undertaking apheresis and evaluation of adequacy for PBSC engraftment. An external quality assurance (EQA) scheme for CD34+ HPC enumeration has been operational in Belgium, Netherlands and Luxemburg (Benelux) since 1995. Within this group, a multicenter survey was held to validate the state-of-the-art methodology, i.e., multiparametric definition of HPC based on light scatter, expression of CD34 and CD45, and counting beads (i.e., 'single platform ISHAGE' method). METHODS: 'Real-time' EQA was used to monitor the application of the single-platform ISHAGE method by 36 participants. Three send-outs of stabilized blood with CD34+ cell counts 35-60 cells/microl were distributed to 36 participants, who were required to assay the samples on three occasions using the standard assay and their local techniques. These results were compared with thosed obtained by 111-116 UK NEQAS participants testing the same specimens. RESULTS: Using the single platform ISHAGE methods, between-laboratory coefficients of variations (CVs) as low as 10% were achieved. Intra-laboratory CVs were < 5% for approximately 50% of the participants. Local single-platform techniques yielded between-laboratory CVs as low as 9% in both Benelux and UK NEQAS cohorts. In contrast, the lowest between-laboratory CVs using dual-platform techniques were 17% (Benelux) and 21% (UK NEQAS), respectively. CONCLUSION: The single-platform ISHAGE method for CD34+ cell enumeration has been validated by an international group of 36 laboratories. The observed varation between laboratories allows a meaningful comparison of CD34+ cell enumeration.

Gas-phase conformers of the [M + 2H](2+) ion of bradykinin investigated by combining high-field asymmetric waveform ion mobility spectrometry, hydrogen/deuterium exchange, and energy-loss measurements.

The continuous separation capability of high-field asymmetric waveform ion mobility spectrometry (FAIMS) was used in combination with complementary techniques for probing biomolecular ions in the gas phase. Gas-phase conformers of the [M + 2H](2+) ion of bradykinin were examined using a combination of FAIMS, H/D exchange, and energy-loss measurements. When FAIMS data and H/D exchange data were analyzed separately, the presence of only two conformers of the [M + 2H](2+) ion of bradykinin could be detected. However, in an experiment in which FAIMS and H/D exchange were combined, at least four different conformers of the gas-phase [M + 2H](2+) ion of bradykinin were detected, including one of very low abundance. Cross sections calculated for the four conformers, based on energy-loss measurements, were 250, 240, 250, and 244 A(2), in order of decreasing abundance.

UK NEQAS for leucocyte immunophenotyping: the first 10 years.

In the past decade, cellular immunophenotyping has become a new discipline in diagnostic haematology and immunology, and is invaluable in the rapid diagnosis of leukaemia and monitoring disease progression in human immunodeficiency virus infected individuals. The introduction of bench top flow cytometers has meant that immunophenotyping is now also used for the quantitation of CD34(+) peripheral blood stem cells (PBSCs) to ensure the correct timing and adequacy of haematopoietic progenitor cell harvests. Furthermore, flow cytometry has become an important tool for the counting of leucocytes in blood components after leucocyte depletion. Because this new discipline is now such a major diagnostic and prognostic tool in the clinical arena, its use must be subject to both internal and external quality control. Such a requirement was first recognised as early as 1986 when an Inter-Regional Quality Assessment Scheme (IRQAS) was initiated for laboratories that undertook the immunocytochemical diagnosis of leukaemia using the alkaline phosphates anti-alkaline phosphatase technique. This programme began with around 25 UK laboratories. In 1990, after the introduction of two more programmes (one for leukaemia diagnosis using UV microscopy and latterly flow cytometry, and one for the enumeration of CD4(+) T cells) the IRQAS achieved UK National External Quality Assessment Scheme (UK NEQAS) status and changed its title to UK NEQAS for Leucocyte Immunophenotyping. In the past decade the once small IRQAS programme has evolved into the largest international scheme of its kind, providing EQA to over 650 laboratories world wide for leukaemia immunophenotyping, lymphocyte subset analysis, PBSCs, and more recently low level leucocyte counting. Over the years, this EQA programme has highlighted important problems, such as the inappropriate use of fluorochromes and antibody titre, and the identification of effective gating strategies, all of which have contributed directly to the high interlaboratory variations seen in cellular immunophenotyping. Furthermore, particularly in absolute counting of lymphocyte subsets, PBSCs, and the enumeration of low numbers of leucocytes, UK NEQAS for Leucocyte Immunophenotyping programmes have been instrumental in highlighting the differences that occur between single and dual platform flow cytometric technologies. As a result of these findings, UK NEQAS for Leucocyte Immunophenotyping has helped to reduce the variation seen on an interlaboratory basis and enabled greater standardisation both in the UK and internationally. These advances have been attributable to the development, by UK NEQAS for Leucocyte Immunophenotyping, of a unique whole blood stabilising process that ensures the retention of the physical characteristics (both light scatter and antigenic profile) required of cells to ensure successful cellular immunophenotyping. This major technological advancement has enabled the distribution of specimens for EQA purposes on a global scale that have minimal matrix effect and behave in a manner identical to fresh blood for several months after stabilisation.

Mesenchymal factor bone morphogenetic protein 4 restricts ductal budding and branching morphogenesis in the developing prostate.

The budding of the urogenital sinus epithelium into the surrounding mesenchyme signals the onset of prostate morphogenesis. The epithelial and mesenchymal factors that regulate ductal budding and the ensuing process of ductal growth and branching are not fully known. We provide evidence that bone morphogenetic protein 4 (BMP4) is a mesenchymal factor that regulates ductal morphogenesis. The Bmp4 gene was most highly expressed in the male urogenital sinus from embryonic day 14 through birth, a period marked by formation of main prostatic ducts and initiation of ductal branching. From an initial wide distribution throughout the prostatic anlage of the urogenital sinus, Bmp4 expression became progressively restricted to the mesenchyme immediately surrounding the nascent prostatic ducts and branches. Exogenous BMP4 inhibited epithelial cell proliferation and exhibited a dose-dependent inhibition of ductal budding in urogenital sinus tissues cultured in vitro. Adult Bmp4 haploinsufficient mice exhibited an increased number of duct tips in both the ventral prostate and coagulating gland. Taken together, our data indicate that BMP4 is a urogenital sinus mesenchymal factor that restricts prostate ductal budding and branching morphogenesis.

Plasma N-terminal pro BNP and cardiotrophin-1 are elevated in aortic stenosis.

BACKGROUND: Echocardiography with Doppler examination of the aortic valve provides a very accurate assessment of the transvalvular gradient and is used to monitor progression of aortic stenosis (AS). Plasma brain natriuretic peptide (BNP) has been shown to correlate with end-systolic wall stress in patients with AS. AIM: We hypothesized that plasma N-terminal proBNP (NT proBNP) and a newly identified cytokine cardiotrophin-1 (CT-1), which has been shown to stimulate BNP production at a transcriptional level are elevated in patients with AS and correlate to the maximum trans-valvular aortic pressure gradient (TVPG). METHOD: We compared plasma NT proBNP and CT-1 in 15 AS patients [five males, mean age 79 years [range 60-94], mean TPVG 39.3 mmHg (20-100)] with 10 controls (five male, mean age 68 years [56-79]). Results are expressed as mean [ranges] and comparisons were by the Mann-Whitney test. RESULTS: NT proBNP levels were elevated in AS patients [252.9 fmol/ml (79.2-541.8)] when compared with the controls (157.2 fmol/ml [104.7-236.9], P<0.005). Also CT-1 levels were elevated in AS patients (57.3 fmol/ml [33-86.3] when compared with the controls [28.3 fmol/ml (6.9-48.3), P<0.0005]. Both NT proBNP and CT-1 levels were correlated to the TVPG (r=0.53 and r=0.65, P<0.05 and P=0.009, respectively). On best subset analysis the strongest correlate with TVPG was CT-1 (R2=38%). The addition of NT proBNP did not improve diagnostic accuracy (R2=39%). CONCLUSION: These results suggest NT proBNP and CT-1 levels increase in proportion to the TVPG and could potentially be used to monitor progression of disease non-invasively. These markers may also be useful to identify the optimum time for surgery in AS.

Detection of microcystins using electrospray ionization high-field asymmetric waveform ion mobility mass spectrometry/mass spectrometry.

A combination of electrospray ionization, high-field asymmetric waveform ion mobility spectrometry, and mass spectrometry (ESI-FAIMS/MS) was used to analyze standard solutions of microcystins-LR, -RR, and -YR. The ability of FAIMS to separate ions in the gas phase reduced the amount of background in the mass spectrum without compromising the absolute signal for these microcystins. This reduction in background resulted in a ten-fold improvement in the signal-to-background ratio over conventional ESI-MS. Detection limits, using direct infusion, were determined to be 4, 2, and 1 nM for microcystins-LR, -RR, and -YR, respectively.

Standardization of lymphocyte antibody binding capacity - a multi-centre study.

As quantitative flow cytometry is being increasingly used to characterize non-malignant and malignant disorders, interlaboratory standardization becomes an important issue. However, the lack of standardized methods and process controls with predefined antibody binding capacity values, limits direct interlaboratory comparison. The present study has addressed these issues using a stable whole blood product and a standardized antigen quantification protocol. It was demonstrated that: (i) a standard technical protocol can result in a high degree of interlaboratory concordance; (ii) interlaboratory variation of less than 12% can be achieved for CD4 antibody binding capacity values; and (iii) stable whole blood can be used as a process control with predefined antibody binding capacity values. Furthermore, using such an approach, a normal range was established for CD3, CD4 CD8 and CD19. These antigens appear to be expressed in a hierarchical manner, a factor that could be used as a procedural quality control measure.