In-house Methods Research Articles

The impact of porcine reproductive and respiratory syndrome virus genetic heterogeneity on molecular assay performances

The remarkable economic losses due to porcine reproductive and respiratory syndrome (PRRS) have stated the control and eradication of this disease is one of the main issues of swine modern farming. The limited cross-protection of vaccine-induced immunity compelled the adoption of strict biosecurity measures that must be associated with the prompt diagnosis of infection. In our study four RT-PCR methods, a RT-PCR, a SYBR Green I and two hydrolysis probes, were compared to evaluate their respective benefits and disadvantages. One hundred and seventy samples originating from 50 farms located in northern Italy were tested with all assays and performances were evaluated using a Bayesian approach to deal with the absence of a Gold Standard. Sequencing the complete of ORF7, the segment targeted by all methods, allowed a gain of insight into the genetic variability of Italian strains and to investigate the role of mismatches on assay sensitivity. Our study evidenced that methods based only on primers-genome interaction better tolerate PRRSV genetic variability, demonstrating a greater sensitivity (Se): SYBR Green I (Se=98.4%) and RT-PCR (Se=99%) outperform both in-house (Se=71.4%) and commercial (Se=91.7%) probe-based methods. On the other hand, probe-based assays allowed an easier genotyping of PRRSV strains and implementation of the internal control system (IC). Phylogenetic analysis allowed demonstration of a presence of two clades circulating continuously in northern Italy since 1996, when their probable ancestors were collected.

Standardizing Leucocyte PNH clone detection: An international study.

Background: Consensus and Practical Guidelines for robust high-sensitivity detection of glycophosphatidylinostitol (GPI)-deficient structures on Red Blood Cells (RBCs) and White Blood Cells (WBCs) in Paroxysmal Nocturnal Hemoglobinuria (PNH) were recently published. Methods: UK NEQAS LI issued 3 stabilized samples manufactured to contain no PNH cells (normal), approximately 0.1% and 8% PNH leucocyte populations, together with instrument-specific SOPs and pre-titered antibody cocktails to 19 international laboratories experienced in PNH testing. Samples were tested using both standardized protocol/reagents and in-house protocols. Additionally, samples were issued to all participants in the full PNH EQA programmes. Results: Expert laboratory results showed no difference in PNH clone detection rates when using standardized and their 'in-house' methods though lower variation around the median was found for the standardized approach compared to in-house methods. Neutrophil analysis of the sample containing an 8% PNH population, for example, showed an interquartile range of 0.48% with the standardized approach compared with 1.29% for in-house methods. Results from the full EQA group showed the greatest variation with an inter-quartile range of 1.70 and this was demonstrated to be significantly different (P<0.001) to the standardized cohort. Conclusions: The results not only demonstrate that stabilized whole PNH blood samples are suitable for use with currently recommended high-sensitivity reagent cocktails/protocols but also highlight the importance of using carefully selected conjugates alongside the standardized protocols. While much more variation was seen amongst the full UK NEQAS LI EQA group, the standardized approach lead to reduced variation around the median even for the experienced laboratories. © 2014 Clinical Cytometry Society.

Evaluation of concentration and purity of DNA extracted from mouthwash, saliva and buccal cytobrush samples

IntroductionNon-invasive sampling has been highlighted as an alternative sampling for molecular analysis of diseases. Various methods of sampling and DNA isolation have been introduced. This study includes collection of mouthwash, saliva and buccal swab/cytobrush sampling, and extraction using conventional methods. ObjectiveTo assess the DNA quantity and quality by evaluating the concentration and purity of DNA isolated from mouthwash, saliva and buccal cytobrush using in-house DNA extraction methods. MethodsMouthwash, saliva and buccal cytobrush samples were collected from paediatric patients attending monthly blood transfusions in University Malaya Medical Centre. The samples were extracted using alkaline lysis method and purified with phenol:chloroform. The DNA purity and concentration was measured by spectrophotometry. Mean and standard error of mean were calculated and analysed for significance in difference. Results & DiscussionThe DNA with highest purity (1.743 ± 0.031) was obtained from mouthwash samples while DNA with highest concentration (0.796 ± 0.095 μg/μL) was obtained from saliva. Buccal cytobrush samples produced DNA with high purity comparable to mouthwash samples, however, at a lower concentration (0.162 ± 0.041 μg/μL). ConclusionHigh quality DNA can be obtained from mouthwash, saliva and buccal cytobrush samples. Saliva samples may serve as the best alternative sampling for extraction of genomic DNA for its high concentration and acceptable purity.

Sickle Cell Solubility Test: Evaluation of an In-House Method

This study aims to assess the performance of an in-house sickle cell solubility test (SCT) and compare it against data published on other in-house and commercial methods. Also assessed are the effects of possible interference due to haemoglobin (Hb) level, Hb F and Hb S levels, as well as lipaemia, icterus and haemolysis index. A total of 1030 patient samples were tested by the SCT and subsequently confirmed by high-performance liquid chromatography (HPLC). Seventy-five (7.3%) samples revealed a positive SCT whereas 955 (92.7%) were negative. The sensitivity and specificity of the method was 100% and it was shown to detect low levels of Hb S (12.1%). These data show that the authors’ in-house SCT method has an excellent performance against other documented methods (including some commercial kits), which is probably explained by its robustness against low levels of Hb S and interfering substances such as lipaemia. The authors believe this shows that in-house tests are able to produce highly reliable results comparable to, if not better than, certain commercially available CE-marked kits.

The Impact Of Dietary Vitamin K On The Pharmacological Activity Of FXa Inhibitor, Rivaroxaban, In Man

Introduction/Background Over several decades, warfarin has been used as the drug of choice for prevention and treatment of thromboembolic disorders. The impact of dietary vitamin K intake on anticoagulation response to warfarin is well established. However, the effect of dietary vitamin K on the activity of the novel oral anticoagulants is less well known. Since the activation of both factor X and thrombin is inextricably linked to the vitamin K cycle in the coagulation cascade, there is the possibility that the pharmacological activity of agents that inhibit these proteins could be influenced by alterations in vitamin K availability. Aim To examine the influence of dietary vitamin K intake on the pharmacological activity of rivaroxaban in man. Methods The pharmacological activity of rivaroxaban was evaluated ex-vivo. Thirty one medically stable elderly inpatients (12 males) with a poor dietary intake in terms of calories and nutrients including vitamin K (12 males) and 28 healthy subjects (7 males) with adequate diets were enrolled into the study. An overnight fasted venous blood sample (20 ml) was taken from each subject. The plasma was stored at -80 degree C for the later measurement of the effects of rivaroxaban on clotting times and clotting factor activity. Each subject completed a validated dietary questionnaire for quantification of vitamin K content of foods eaten in the past week. The plasma samples from each subject were incubated with a range of rivaroxaban concentrations (100 - 500 ng/ml) in order to simulate the therapeutic drug plasma concentrations. Normal prothrombin time (PT), modified prothrombin time (mPT), and vitamin K dependent clotting factors activity were measured according to established in-house methods. Results The mean±SD age of elderly patients and healthy subjects was 87±6 and 36±10 years, respectively. Rivaroxaban produced a greater prolongation of both PT (P=0.01) and m-PT (P=0.02) in the plasma of elderly subjects than the younger healthy subjects over the drug concentration range studied. The mean difference in PT between the two groups ranged from 1.8s to 5.0s at 100 ng/ml and 500 ng/ml plasma rivaroxaban concentrations respectively. The mean difference in m-PT between the two groups ranged from 10.0s to 34.0s at 100 ng/ml and 500 ng/ml plasma rivaroxaban concentrations respectively. There was also a greater inhibition of factor Xa (P=0.005) activity by rivaroxaban in the elderly subjects compared to the healthy subjects, whereas factor IXa (P=0.03) activity was significantly inhibited in the healthy subjects compared to the elderly subjects. However, there were no significant differences between the two groups in factors II and VII activity and fibrinogen concentration. The analysis of dietary questionnaires for vitamin K intake and plasma for fasting vitamin K levels is in progress and the results will be presented. Conclusion We have previously demonstrated in a proof-of-concept study in rats that vitamin K deficiency significantly enhanced the pharmacological activity of the prototype DTI, ximelagatran [1]. The present study findings suggest that the anticoagulation response to rivaroxaban may be influenced by poor dietary vitamin K intake. Assessment of whether dietary vitamin K influences anticoagulation response as well as any influence of age, co morbidities and drug intake in patients taking rivaroxaban is warranted. [1] Kamali F, Wood P, Ward A. Vitamin K deficiency amplifies anticoagulation response to ximelagatran: possible implications for direct thrombin inhibitors and their clinical safety. Annals of Hematology 2009; 88:141-149. Disclosures: No relevant conflicts of interest to declare.

Solution casting of chitosan membranes for in vitro evaluation of bioactivity

BackgroundConsiderable research is focusing on the surface modification of titanium implants for the treatment of orthopaedic tissue injuries to increase the success of orthopaedic fixations. Chitosan is one of the natural materials under investigation based on several favourable properties. Numerous techniques have been described for the preparation of chitosan membranes, including solution casting methods for the investigation of bioactivity before applying coatings onto potential titanium implants. Solution casting enables the easy in-house evaluation of chitosan membranes and allows for the selection of promising chitosan materials.ResultsWe present a method for the standardized and easily applied preparation of chitosan membranes by solution casting. This protocol is suitable for chitosan materials spanning a wide degree of deacetylation, being derived from different chitin sources and chitosan derivatives with novel properties. We detail the preparation and quality control methods in order to prepare membranes with favourable bioactivity, sustaining cell attachment and proliferation for extended culture periods.ConclusionsThe possibilities associated with the use of chitosan in tissue engineering applications are far from being exhausted and numerous challenges remain prior to successful translation into the clinics. Based on our experience, we have developed simple in-house methods for quality control of homogeneous membrane casting and early prediction of successful experimental outcome.

Validation of a specific prolylcarboxypeptidase activity assay and its suitability for plasma and serum measurements

Prolylcarboxypeptidase (PRCP, EC 3.4.16.2), a lysosomal carboxypeptidase, was discovered 45years ago. However, research has been hampered by a lack of well-validated assays that are needed to measure low activities in biological samples. Two reversed-phase high-performance liquid chromatography (RP-HPLC) methods for quantifying PRCP activity in crude homogenates and plasma samples were optimized and validated. PRCP activity was determined by measuring the hydrolysis of N-benzyloxycarbonyl-l-proline (Z-Pro)-Phe. The enzymatically formed Z-Pro and Phe were measured independently under different HPLC conditions. The in-house methods showed good precision, linearity, accuracy, and specificity. Based on Michaelis–Menten constants, Z-Pro-Phe was chosen over Z-Pro-Ala as the substrate of preference. Cross-reactivity studies with dipeptidyl peptidases (DPPs) 2, 4, and 9 and prolyl oligopeptidase (PREP) confirmed the specificity of the PRCP activity assay. The average PRCP activity in plasma and serum of 32 healthy individuals was found to be 0.65±0.02 and 0.72±0.03U/L, respectively. Both methods can be used to measure PRCP activity specifically in different biological samples and are well suited to evaluate PRCP inhibitors. These well-validated methods are valuable tools for studying PRCP’s role in cardiovascular diseases, stroke, inflammation, and metabolic syndrome.

The Soluble Receptor for Vitamin B12 Uptake (sCD320) Increases during Pregnancy and Occurs in Higher Concentration in Urine than in Serum

BackgroundCellular uptake of vitamin B12 (B12) demands binding of the vitamin to transcobalamin (TC) and recognition of TC-B12 (holoTC) by the receptor CD320, a receptor expressed in high quantities on human placenta. We have identified a soluble form of CD320 (sCD320) in serum and here we present data on the occurrence of this soluble receptor in both serum and urine during pregnancy.MethodsWe examined serum from twenty-seven pregnant women (cohort 1) at gestational weeks 13, 24 and 36 and serum and urine samples from forty pregnant women (cohort 2) tested up to 8 times during gestational weeks 17-41. sCD320, holoTC, total TC and complex formation between holoTC and sCD320 were measured by in-house ELISA methods, while creatinine was measured on the automatic platform Cobas 6000. Size exclusion chromatography was performed on a Superdex 200 column.ResultsMedian (range) of serum sCD320 increased from 125 (87-839) pmol/L (week 15) to reach a peak value of 199 (72-672) pmol/L (week 35) then dropped back to its baseline level just before birth (week 40). Around one third of sCD320 was precipitated with holoTC at all-time points studied. The urinary concentration of sCD320 was around two fold higher than in serum. Urinary sCD320/creatinine ratio correlated with serum sCD320 and reached a peak median level of 53 (30–101) pmol/mmol creatinine (week 35). sCD320 present in serum and urine showed the same elution pattern upon size exclusion chromatography.ConclusionWe report for the first time that sCD320 is present in urine and in a higher concentration than in serum and that serum and urine sCD320 increase during pregnancy. The high urinary concentration and the strong correlation between urinary and serum sCD320 suggests that sCD320 is filtered in the kidney.

Validation and comparison of a sandwich ELISA, two competitive ELISAs and a real-time PCR method for the detection of lupine in food

Methods applied in food allergen analysis should be specific, sensitive and applicable to both raw and highly processed foods. The performance of the most commonly used methods, ELISA and real-time PCR, may, however, be influenced by food processing steps, e.g., heat treatment. The present study compares the applicability of four in-house developed methods, one sandwich ELISA, two competitive ELISAs and a real-time PCR method, for the detection of lupine in four different food matrices, comprising bread, biscuits, rice patties and noodles. In order to investigate the influence of food processing on the detectability, not only the heat treated model foods but also the corresponding doughs were analysed. The sandwich ELISA proved to be the most sensitive method. The LOD was found to be 10ppm lupine, independent from the food matrix and independent if the dough or the heat treated food was analysed. In addition, the methods were applied to the analysis of commercial foodstuffs differing in their labelling.

PCR revisited: a case for revalidation of PCR assays for microorganisms using identification ofCampylobacterspecies as an exemplar

We re-examined the sensitivity and specificity of 31 PCR assays (including four commercially available and developed in-house methods) for the identification of Campylobacter species, with particular reference to taxa described since 2004, which are closely related to C. jejuni and C. coli, the pathogenic species of most interest. Each of the assays was used by at least one of the participating nine laboratories in eight countries. The sensitivity and specificity of these PCR assays examined varied considerably and ranged from 100% to 0% for sensitivity and 100% to 55% for specificity. None of the three assays examined for C. lari were successful in detecting all strains of this species, possibly reflecting its complex taxonomy. A number of assays for C. jejuni, C. coli, and a subgroup of enteropathogenic campylobacters, were found to yield false positive results for Campylobacter species described since PCR tests were reported, including C. cuniculorum, C. subantarcticus, C. peloridis and C. volucris. Our study supports the need for attention to detail in initial PCR assay design and evaluation, and also for on-going revalidation of laboratory assays to ensure that diagnoses are correct. Recommendations to guide the revalidation process are presented.

Vitamin B1 and B6 method harmonization: Comparison of performance between laboratories enrolled in the RCPA Quality Assurance Program

ObjectivesThe RCPA Quality Assurance Program (RCPA QAP) offers monthly proficiency testing for vitamins A, B1, B6, β-carotene, C and E to laboratories worldwide. A review of the results submitted for the whole blood vitamin B1/B6 sub-program revealed a wide dispersion. Here we describe the results of a methodology survey for vitamins B1 and B6. Design and methodsA questionnaire was sent to thirteen laboratories. Eleven laboratories were returning QAP results for vitamin B1 (thiamine diphosphate) and five were returning results for vitamin B6 (pyridoxal-5-phosphate). ResultsAll nine respondents provided a clinical service for vitamins B1 and B6. HPLC with fluorescence detection was the most common method principle. For vitamin B1, six respondents used a commercial assay whilst three used in-house methods; whole blood was the matrix for all. For vitamin B6, five respondents used commercial assays and four used in-house assays. The choice of matrix for vitamin B6 varied with three respondents using whole blood and five using plasma for analysis. Sample preparation incorporated protein precipitation and derivatization steps. An internal standard was employed in sample preparation by only one survey respondent. ConclusionsThe immediate result of this survey was the incorporation of plasma vitamin B6 into the RCPA QAP vitamin program. The absence of an internal standard in current vitamin B1 and B6 assays is a likely contributor to the wide dispersion of results seen in this program. We recommend kit manufacturers and laboratories investigate the inclusion of internal standards to correct the variability that may occur during processing.

Clinical Management of Drug-Resistant Tuberculosis in Resource Constrained Settings

Drug-resistant forms of tuberculosis (TB), particularly multi- and extensively drug-resistant TB, represent an important obstacle to global control of the disease. Recently, new drugs, repurposed drugs, and new drug combinations have been evaluated, with a number showing promise for the treatment of drug-resistant TB. Additionally, a range of methods for accelerating mycobacterial culture, identification, and drug susceptibility testing have been developed, and several in-house and commercial genotyping methods for speeding drug resistance detection have become available. Despite these significant achievements in drug development and diagnostics, drug-resistant TB continues to be difficult to diagnose and treat. Significant international efforts are still needed, especially in the field of clinical and operational research, to translate these encouraging developments into effective patient cure and make them readily available to resource-constrained settings, where they are most needed.

Direct urine polymerase chain reaction for chlamydia and gonorrhoea: a simple means of bringing high-throughput rapid testing to remote settings?

Background Rapid point-of-care tests (POCTs) for chlamydia (Chlamydia trachomatis) and gonorrhoea (Neisseria gonorrhoeae) have the potential to confer health benefits in certain populations even at moderate sensitivities; however, suitable POCTs for these organisms are currently lacking. In this study, we investigated the use of direct urine polymerase chain reaction (PCR), with the view of implementing a simplified PCR strategy for high-throughput chlamydia and gonorrhoea screening in remote settings. Briefly, a simple dilution of the urine was performed before adding it directly to a real-time PCR reaction. The method was evaluated using 134 stored urine specimens that had been submitted for chlamydia and gonorrhoea testing and had been tested using a commercial C. trachomatis and N. gonorrhoeae PCR method. These included samples that were PCR-positive for chlamydia (n=87), gonorrhoea (n=16) or both (n=2). Direct urine testing was conducted using previously described in-house real-time PCR methods for C. trachomatis and N. gonorrhoeae as well as for recognised N.gonorrhoeae antimicrobial resistance mechanisms. The overall sensitivities and specificities of the direct urine PCR were 78% and 100% for chlamydia, and 83% and 100% for gonorrhoea. N.gonorrhoeae penicillin and quinolone resistance mechanisms were characterised in 14 of the 18 N. gonorrhoeae-positive samples. The results of this study show that the simplified PCR strategy may be a feasible approach for rapid screening and improving chlamydia and gonorrhoea treatment in remote settings.

English

There are major concerns worldwide regarding sexually transmitted infections (STI), bacterial vaginosis (BV), and candidiasis as a major cause of morbidity as they result in significant health and economic consequences, particularly in developing countries. This study was intended to obtain information about the prevalence of these pathologies in women considered to be at low risk using both traditional and&nbsp;in-house&nbsp;NAAT methods. Cervical and vaginal samples were collected all volunteers signed an informed consent form and completed a survey. BV, trichomoniasis, candidiasis, genital mycoplasmas colonization and&nbsp;Chlamydia trachomatis&nbsp;or&nbsp;Neisseria gonorrhoeae&nbsp;cervicitis were diagnosed. Candidiasis and abnormal vaginal flora associated with BV were very frequent. The high colonization with micoplasmas was detected.&nbsp;C. trachomatis&nbsp;cervicitis was found in 10.67% from which a third of the cases were of asymptomatic woman. No cases of gonorrhoea or trichomoniasis were diagnosed.&nbsp;In house&nbsp;NAAT&rsquo;s used seems to viable tools for the cheap and reliable test for the diagnosis of gonorrhoea and chlamydial infections. Increased awareness of the importance of protected sexual intercourse is imperative to prevent the transmission of sexually transmitted. Further studies for a comprehensive understanding of the rates of these infections in Mexican women are necessary and should be an impulse for make community-based assessment of STI and RTI. &nbsp; Key words:&nbsp;Bacterial vaginosis, candidosis, STI, cervicitis, gonorrhoea.

Diagnostic Assays for Crimean-Congo Hemorrhagic Fever

Crimean-Congo hemorrhagic fever (CCHF) is a highly contagious viral tick-borne disease with case-fatality rates as high as 50%. We describe a collaborative evaluation of the characteristics, performance, and on-site applicability of serologic and molecular assays for diagnosis of CCHF. We evaluated ELISA, immunofluorescence, quantitative reverse transcription PCR, and low-density macroarray assays for detection of CCHF virus using precharacterized archived patient serum samples. Compared with results of local, in-house methods, test sensitivities were 87.8%-93.9% for IgM serology, 80.4%-86.1% for IgG serology, and 79.6%-83.3% for genome detection. Specificity was excellent for all assays; molecular test results were influenced by patient country of origin. Our findings demonstrate that well-characterized, reliable tools are available for CCHF diagnosis and surveillance. The on-site use of such assays by health laboratories would greatly diminish the time, costs, and risks posed by the handling, packaging, and shipping of highly infectious biologic material.

In-house preparation of hydrogels for batch affinity purification of glutathione S-transferase tagged recombinant proteins

BackgroundMany branches of biomedical research find use for pure recombinant proteins for direct application or to study other molecules and pathways. Glutathione affinity purification is commonly used to isolate and purify glutathione S-transferase (GST)-tagged fusion proteins from total cellular proteins in lysates. Although GST affinity materials are commercially available as glutathione immobilized on beaded agarose resins, few simple options for in-house production of those systems exist. Herein, we describe a novel method for the purification of GST-tagged recombinant proteins.ResultsGlutathione was conjugated to low molecular weight poly(ethylene glycol) diacrylate (PEGDA) via thiol-ene “click” chemistry. With our in-house prepared PEGDA:glutathione (PEGDA:GSH) homogenates, we were able to purify a glutathione S-transferase (GST) green fluorescent protein (GFP) fusion protein (GST-GFP) from the soluble fraction of E. coli lysate. Further, microspheres were formed from the PEGDA:GSH hydrogels and improved protein binding to a level comparable to purchased GSH-agarose beads.ConclusionsGSH containing polymers might find use as in-house methods of protein purification. They exhibited similar ability to purify GST tagged proteins as purchased GSH agarose beads.

Evaluation of indirect immunofluorescence assay in patients with autoimmune diseases

Antinuclear antibodies (ANA) are the hallmark of autoantibody production in autoimmune disease. ANA are directed against components of the cell nuclei such as DNA, histones, nucleoli and ribonucleoproteins. ANA detected by indirect immunofluorescence (IIF) is the method of choice and is regarded as the gold standard technique. 100 patients with autoimmune disease attending a tertiary care hospital at Chennai, India and ten healthy controls were included in the study. Detailed clinical, serological and biochemical analysis were done. Blood samples were analyzed for ANA by IIF using in-house mouse liver and in-house HEp-2 cell line substrate. The cells showing apple green fluorescence and a definable nuclear staining pattern were taken as positive and the intensity of fluorescence graded from + to ++++ in serial dilutions of the sera. IIF patterns like homogeneous (solid) rim (peripheral), speckled and nucleolar patterns were observed. ANA positivity by IIF for SLE is 92.7 and 97.4% on mouse liver and HEp-2 cells, respectively. Specificity of ANA by IIF is 80 and 90% for mouse liver and HEp-2 cell substrate, respectively. Although, many commercial assays are available for the detection of ANA, IIF by in-house methods still holds good and is justified in this research work. Key words: Autoantibodies, indirect immunofluorescence, antinuclear antibodies, systemic lupus erythematosus (SLE).

Firms' Choice of a Social Impact Measurement Method to Evaluate Base of the Pyramid Strategies: A Decision Support Framework

The need to measure the social impact of Bottom of the Pyramid (BOP) strategies has become a consensus in the field, but identifying how to approach such an undertaking remains a challenge for firms. This paper builds on a research collaboration with a Multinational Corporation (MNC) engaged in housing programs at the BOP. This paper proposes a framework to assist in the firm’s choice of a social impact assessment method according to its objectives. Three categories of social impact evaluation methods are identified: ready-to-use tools, in-house methods and statistical methods. The different objectives that a firm may set for its impact assessment are accountability and communication, program improvement in the short run, and innovation in the long run. We propose to link each of the objectives to one method category and thereby facilitate the MNC’s choice of a social impact assessment method.

SU-E-T-421: 6MV Radiation Small Field Dose: Off Axis and Penumbra Effects, a Study with Radiochromic Film.

To accurately determine relative dose factor scaling, tissue depth relation, and penumbra details of small 6MV radiation fields with an emphasis on far-off-central-axis fields for various MLC designs -subject matter important in the context of SRS, FSRT, SBRT, and IMRT. Radiochromic film designed for quantitative dosimetry, EBT3™ film (Ashland Inc.) can be reliably used as a relative dosimeter to within 1% accuracy if one controls for both film and flatbed scanner variation. This enabled a study of the fine detail of small radiation fields and the impact upon these small dose profiles by different MLC designs both on central axis and off. In particular, the impact of leaf-end penumbra from bifocal MLC Optifocus (Siemens) and Synergy Beam Modulator (Elekta) is examined.EBT3 films were placed at 100cm SAD in solid water phantom and irradiated to 200 MU at various depths 1.5cm - 20cm and for various off-axis locations. The films were analysed to give the detailed dosimetric profile of each small field and relate them to both a standard reference field (10 × 10)cm2 and to a broadfield flatness/symmetry irradiation (20 × 20)cm2 . Films were scanned at ∼20 hours post irradiation on an Epson 2000 flatbed scanner and analysed using FilmQAPro and in-house methods. As small field size decreases, the field center falls within the penumbra of each MLC edge for megavoltage photon energies. The penumbral effects cause the conventional dosimetric quantity SP to sharply depart from the smooth fieldsize dependence for larger conventional fields. Careful characterization of small field dose and leaf end modeling within a TPS is crucial in both predicting accurate small field dosimetry and off-axis dosimetry.

Evaluation of the performance of selected in-house and commercially available PCR and real-time PCR assays for the detection of Leishmania DNA in canine clinical samples

Protozoa of the genus Leishmania are the causative agents of leishmaniosis. Although the polymerase chain reaction (PCR) has proved very effective in the detection of Leishmania DNA, a standardized method does not exist. In this study we attempt a comparative evaluation between one real time PCR (Method D), two in-house (Methods A and C), and a commercially available PCR assay (Method B) for the detection of Leishmania DNA, in order to support reliable diagnostic investigation of leishmaniosis. This evaluation was performed in regard to relative specificity and sensitivity, minimum detection limit (MDL), repeatability and reproducibility using cultured isolates and clinical samples. All the methods under study produced the expected result with the positive and negative controls. However with regard to clinical samples, Method C showed a statistically significant higher level of positivity. Relative sensitivity and specificity of Methods A, B and D in comparison to C was calculated respectively at 50.7%, 43%, 40%, and 90.8%, 93.4% and 89.5%. The MDL for Methods A–D was defined respectively at 30.7, 5, 3.7, and 5 promastigotes/ml. Repeatability and reproducibility were excellent in all cases with only the exception of Method A regarding reproducibility with a different brand of PCR reagents. The results that were recorded indicate that evaluation of PCR assays before their application for research and clinical diagnosis can provide useful evidence for their reliable application. Within this context the use of internal amplification controls and the confirmation of the specificity of the amplification product is recommended.