Simulation methods in the modelling of bioaffinity assays

Computational model-based simulation methods were developed for the modelling of bioaffinity assays. Bioaffinity-based methods are widely used to quantify a biological substance in biological research, development and in routine clinical in vitro diagnostics. Bioaffinity assays are based on the high affinity and structural specificity between the binding biomolecules. The simulation methods developed are based on the mechanistic assay model, which relies on the chemical reaction kinetics and describes the forming of a bound component as a function of time from the initial binding interaction. The simulation methods were focused on studying the behaviour and the reliability of bioaffinity assay and the possibilities the modelling methods of binding reaction kinetics provide, such as predicting assay results even before the binding reaction has reached equilibrium. For example, a rapid quantitative result from a clinical bioaffinity assay sample can be very significant, e.g. even the smallest elevation of a heart muscle marker reveals a cardiac injury. The simulation methods were used to identify critical error factors in rapid bioaffinity assays. A new kinetic calibration method was developed to calibrate a measurement system by kinetic measurement data utilizing only one standard concentration. A nodebased method was developed to model multi-component binding reactions, which have been a challenge to traditional numerical methods. The node-method was also used to model protein adsorption as an example of nonspecific binding of biomolecules. These methods have been compared with the experimental data from practice and can be utilized in in vitro diagnostics, drug discovery and in medical imaging.

Implementation of a trauma registry in a brazilian public hospital: the first 1,000 patients

OBJECTIVE: Show the steps of a Trauma Registry (TR) implementation in a Brazilian public hospital and evaluate the initial data from the database.METHODS: Descriptive study of the a TR implementation in João XXIII Hospital (Hospital Foundation of the state of Minas Gerais) and analysis of the initial results of the first 1,000 patients.RESULTS: The project was initiated in 2011 and from January 2013 we began collecting data for the TR. In January 2014 the registration of the first 1000 patients was completed. The greatest difficulties in the TR implementation were obtaining funds to finance the project and the lack of information within the medical records. The variables with the lowest completion percentage on the physiological conditions were: pulse, blood pressure, respiratory rate and Glasgow coma scale. Consequently, the Revised Trauma Score (RTS) could be calculated in only 31% of cases and the TRISS methodology applied to 30.3% of patients. The main epidemiological characteristics showed a predominance of young male victims (84.7%) and the importance of aggression as a cause of injuries in our environment (47.5%), surpassing traffic accidents. The average length of stay was 6 days, and mortality 13.7%.CONCLUSION: Trauma registries are invaluable tools in improving the care of trauma victims. It is necessary to improve the quality of data recorded in medical records. The involvement of public authorities is critical for the successful implementation and maintenance of trauma registries in Brazilian hospitals.

Understanding the in vitro dissolution rate of glasses with respect to future clinical applications

Glass is a unique material with a long history. Several glass products are used daily in our everyday life, often unnoticed. Glass can be found not only in obvious applications such as tableware, windows, and light bulbs, but also in tennis rackets, windmill turbine blades, optical devices, and medical implants. The glasses used at present as implants are inorganic silica-based melt-derived compositions mainly for hard-tissue repair as bone graft substitute in dentistry and orthopedics. The degree of glass reactivity desired varies according to implantation situation and it is vital that the ion release from any glasses used in medical applications is controlled. Understanding the in vitro dissolution rate of glasses provides a first approximation of their behavior in vivo. Specific studies concerning dissolution properties of bioactive glasses have been relatively scarce and mostly concentrated to static condition studies. The motivation behind this work was to develop a simple and accurate method for quantifying the in vitro dissolution rate of highly different types of glass compositions with interest for future clinical applications. By combining information from various experimental conditions, a better knowledge of glass dissolution and the suitability of different glasses for different medical applications can be obtained. Thus, two traditional and one novel approach were utilized in this thesis to study glass dissolution. The chemical durability of silicate glasses was tested in water and TRIS-buffered solution at static and dynamic conditions. The traditional in vitro testing with a TRISbuffered solution under static conditions works well with bioactive or with readily dissolving glasses, and it is easy to follow the ion dissolution reactions. However, in the buffered solution no marked differences between the more durable glasses were observed. The hydrolytic resistance of the glasses was studied using the standard procedure ISO 719. The relative scale given by the standard failed to provide any relevant information when bioactive glasses were studied. However, the clear differences in the hydrolytic resistance values imply that the method could be used as a rapid test to get an overall idea of the biodegradability of glasses. The standard method combined with the ion concentration and pH measurements gives a better estimate of the hydrolytic resistance because of the high silicon amount released from a glass. A sensitive on-line analysis method utilizing inductively coupled plasma optical emission spectrometer and a flow-through micro-volume pH electrode was developed to study the initial dissolution of biocompatible glasses. This approach was found suitable for compositions within a large range of chemical durability. With this approach, the initial dissolution of all ions could be measured simultaneously and quantitatively, which gave a good overall idea of the initial dissolution rates for the individual ions and the dissolution mechanism. These types of results with glass dissolution were presented for the first time during the course of writing this thesis. Based on the initial dissolution patterns obtained with the novel approach using TRIS, the experimental glasses could be divided into four distinct categories. The initial dissolution patterns of glasses correlated well with the anticipated bioactivity. Moreover, the normalized surface-specific mass loss rates and the different in vivo models and the actual in vivo data correlated well. The results suggest that this type of approach can be used for prescreening the suitability of novel glass compositions for future clinical applications. Furthermore, the results shed light on the possible bioactivity of glasses. An additional goal in this thesis was to gain insight into the phase changes occurring during various heat treatments of glasses with three selected compositions. Engineering-type T-T-T curves for glasses 1-98 and 13-93 were stablished. The information gained is essential in manufacturing amorphous porous implants or for drawing of continuous fibers of the glasses. Although both glasses can be hot worked to amorphous products at carefully controlled conditions, 1-98 showed one magnitude greater nucleation and crystal growth rate than 13-93. Thus, 13-93 is better suited than 1-98 for working processes which require long residence times at high temperatures. It was also shown that amorphous and partially crystalline porous implants can be sintered from bioactive glass S53P4. Surface crystallization of S53P4, forming Na2O∙CaO∙2SiO2, was observed to start at 650°C. The secondary crystals of Na2Ca4(PO4)2SiO4, reported for the first time in this thesis, were detected at higher temperatures, from 850°C to 1000°C. The crystal phases formed affected the dissolution behavior of the implants in simulated body fluid. This study opens up new possibilities for using S53P4 to manufacture various structures, while tailoring their bioactivity by controlling the proportions of the different phases. The results obtained in this thesis give valuable additional information and tools to the state of the art for designing glasses with respect to future clinical applications. With the knowledge gained we can identify different dissolution patters and use this information to improve the tuning of glass compositions. In addition, the novel online analysis approach provides an excellent opportunity to further enhance our knowledge of glass behavior in simulated body conditions.

Sotilaslentäjän fyysinen suorituskyky sekä työperäiset tuki- ja liikuntaelinoireet

Kirjallisuudesta välittyy useitten vuosikymmenten ajalta tietämys sotilaslentämisen fyysisestä kuormittavuudesta. G-voimista aiheutuva kuormittuminen näyttää johtavan joko akuutisti tai pitkäaikaisesti lentäjän tuki- ja liikuntaelimistön toimintakykyä alentaviin ongelmiin. Erityisesti on selvitetty niskan alueen työperäisten ongelmien syntyä, jolloin on havaittu lentotoiminnan fyysisen kuormittavuuden johtavan ennenaikaiseen rakenteelliseen rappeumaan, haittaa aiheuttavan oireen lisäksi. Kansainvälisen kirjallisuuden mukaan ammatista johtuvista eli työperäisistä oireista kärsii vähintään 2/3 kaikista sotilaslentäjistä. Tietyin edellytyksin lentäjien kaularangan alueen rappeuma on Suomessa hyväksytty ammattitaudiksi vuodesta 1995 alkaen. On arveltu, että hyvästä fyysisestä suorituskyvystä olisi apua tuki- ja liikuntaelin (TULE)-oireilun ennaltaehkäisemisessä ja toimintakyvyn ylläpitämisessä. Tutkimusnäyttö tästä on lentäjien osalta ollut toistaiseksi erittäin niukkaa. Tämän tutkimuksen tavoitteena oli selvittää suomalaisten sotilaslentäjien työperäisen TULE-oireilun esiintyvyyttä, oireista koetun haitan tasoa, lentäjien fyysisen kunnon tasoja virkauran aikana ja näitten kaikkien välisiä yhteyksiä sekä työperäisen TULE-oireen merkitystä sotilaan toimintakykyyn. Tutkimus jakautui kahteen osaan. Poikkileikkauksena lentotoimintaperäisiä TULE-oireita kartoitettiin kyselytutkimuksella, johon vastasi vuositarkastuksen yhteydessä 267 lentäjää vuosina 2004-2005. Joukosta poimittiin ne 195 lentäjää, jotka olivat suorittaneet yleissotilaalliset kuntotestit puolen vuoden sisällä kyselyyn vastaamisesta, ja mitatut testitulokset yhdistettiin kyselytutkimusaineistoon. Tässä aineistossa toteutettiin fyysisesti erilailla kuormittuvien lentäjäryhmien välisiä vertailuja fyysisen kunnon, TULE-esiintyvyyden ja koetun haitan suhteen. Poikkileikkausosassa tutkittiin myös lentäjien virkauran aikaisia tasoeroja yleissotilaallisissa kuntotesteissä (n=195) verrattuna muihin suomalaisiin sotilaisiin. Lisäksi (N=289) selvitettiin ilmailulääketieteellisen tarkastuksen yhteydessä mitattuja, ns. ammatillisia fyysisiä erityisominaisuuksia eri ikäluokissa. Pitkittäisosassa seurattiin 67:n Hawk-suihkuharjoituskoneella aloittaneen Ilmavoimien sotilaslentäjien lentouran aikaista lentotoimintaperäisten TULE-oireitten esiintyvyyttä vuosien 1996 ja 2008 välillä. Lisäksi tutkittiin lentäjien kontakteja työterveyshuoltoon, oireen aiheuttamaa lentokelvottomuusaikaa, työn kuormituksen kumulatiivista kertymää lentotuntien lisääntyessä ja TULE-oireiden esiintyvyyden kannalta kriittisiä ajankohtia lentouran aikana. Tulokset osoittivat, että kaikki seurannassa olleet suomalaiset sotilaslentäjät kokivat jonkinasteisen lentotoimintaperäisen TULE-oireen uransa aikana. Niskan ammattitautiluokituksen tasoisen ongelman esiintyvyys oli 4 % koko lentäjäpopulaatiosta ja 10 % suihkuharjoituskonevaiheen jo läpäisseistä, mutta vastaavanlaisia TULE-ongelmia, ilman riittävää näyttöä ammattitaudista, esiintyi lähes joka kolmannella sotilaslentäjällä. Alaselän osalta lentäjät oireilivat lähes samassa määrin, mutta näitä oireita ei toistaiseksi ole mahdollista määrittää ammattitaudiksi. Lentäjät kävivät varsin vähän valittamassa oireistaan työterveyshuoltoon, jossa käytäneen vasta silloin, kun oire jo selvästi heikentää työtehtävissä vaadittavaa toimintakykyä. Merkittävin lentotoimintaperäisten oireitten esiintymisen kasvu ajoittui 200 Hawk-lentotunnin kohdalle, jolloin koneella saavutetaan eräänlainen optimaalinen G-indeksi eli taktisen liikehtelyn G-tasoylitysten vaihtelu. Tämän jälkeen lentäjät ovat erityisen alttiina akuuteille lennonaikaisille TULE-ongelmille. Oireitten esiintyminen kasvoi eksponentiaalisesti noin 600 lentotuntiin asti. Monimuuttujamallien mukaan työperäisen TULE-oireen esiintyvyysriskiä vähensivät alaraajojen hyvä motoriikka, korkeat valintapisteet ja korkea kaulan fleksion voimataso maksimaalisessa isometrisessä testissä. Yleissotilaallisilla kuntotasoilla ei ollut yhteyttä oireiluun, mutta lihaskunnoltaan voimakkaimmat lentäjät kärsivät tilastollisesti merkittävästi vähemmän haittaa lentotoimintaperäisistä TULE-oireistaan. Yleissotilaallisissa kuntotesteissä lentäjät olivat parempia kuin muut suomalaiset sotilaat. Aktiivisimman lentouran aikana, 30-40-vuotiaina, lentäjien fyysinen suorituskyky oli normaaliväestöön nähden vain keskimääräinen ja urheilijoihin nähden keskimääräistä heikompi. Käytännössä lentäjät eivät kyenneet ylläpitämään valintavaiheen fyysistä suorituskykyään edes kadettivaiheen loppuun asti. Huomattavaa oli lisäksi, että aktiivisen lentouran päätyttyä fyysinen kunto näytti jossain määrin palautuvan kohti lähtötasoa lentäjien ikääntymisestä huolimatta. Lentäjien valintavaiheen aikana mitatun fyysisen suorituskyvyn tason säilyminen aktiivisen lentopalveluksen loppuun asti vaatisi lentäjien fyysisen toimintakyvyn ylläpidon ja kehittämisen tehostamista koulutuksen ja työuran eri vaihessa. Tähän tavoitteeseen nähden Ilmavoimien fyysisen kasvatuksen järjestelyt vaikuttivat alimitoitetuilta. Operatiivisesti huolestuttavaa oli Ilmavoimien ohjaajien fyysisen suorituskyvyn heikentyminen silloin, kun heidän taitojensa puolesta olisi pitänyt olla suorituskykyisimpiä taistelutehtäviinsä. Myös lentäjän terveyttä ja toimintakykyä pitäisi pystyä reaaliaikaisemmin seuraamaan koko lentouran aikana. Ilmavoimille suositellaan moniammatillista lähestymistä sotilaslentäjien toimintakyvyn ylläpitämiseen ja terveysriskien hallintaan yhdessä liikunnan, työterveyshuollon, lentoturvallisuusalan ja operatiivisen suunnittelun asiantuntijoitten kanssa. Lisäksi suositellaan avoimempaa ja eettisesti kestävämpää suhtautumista ammattiin liittyvien terveysongelmien kuvaamiseen sekä fyysisen kunnon kysymyksiin jo lentäjien rekrytointivaiheessa.

Evaluation of follicular lymphoid depletion in the Bursa of Fabricius: an alternative methodology using digital image analysis and artificial neural networks

Fifty Bursa of Fabricius (BF) were examined by conventional optical microscopy and digital images were acquired and processed using Matlab® 6.5 software. The Artificial Neuronal Network (ANN) was generated using Neuroshell® Classifier software and the optical and digital data were compared. The ANN was able to make a comparable classification of digital and optical scores. The use of ANN was able to classify correctly the majority of the follicles, reaching sensibility and specificity of 89% and 96%, respectively. When the follicles were scored and grouped in a binary fashion the sensibility increased to 90% and obtained the maximum value for the specificity of 92%. These results demonstrate that the use of digital image analysis and ANN is a useful tool for the pathological classification of the BF lymphoid depletion. In addition it provides objective results that allow measuring the dimension of the error in the diagnosis and classification therefore making comparison between databases feasible.

Polymerase chain reaction for the diagnosis of bovine genital campylobacteriosis

Bovine genital campylobacteriosis is a common venereal disease of cattle; the prevalence of this disease can be underestimated mostly because of the nature of the etiological agent, the microaerobic Campylobacter fetus subspecies venerealis. The purpose of the current study was to evaluate the utilization of polymerase chain reaction (PCR) in the diagnosis of genital campylobacteriosis in samples obtained from bull prepuce aspirate, cow cervical mucus, and abomasum contents of aborted fetuses, collected into enrichment medium. Five different DNA extraction protocols were tested: thermal extraction, lysis with proteinase K, lysis with guanidine isothiocyanate, lysis with DNAzol, and lysis with hexadecyltrimethylammonium bromide (CTAB). The specificity, sensitivity, and technical application of the PCR assay were also evaluated with clinical samples and compared to bacterial isolation by standard culture. DNA extraction by the CTAB protocol provided better results in PCR, and it was able to detect 63 colony-forming units per ml of C. fetus. Out of 277 clinical samples tested, 68 (24%) were positive for Campylobacter fetus using PCR, while only 8 (2.8%) of the samples were positive by bacterial isolation in solid medium, proving the superiority of the PCR technique when compared to the standard isolation method, and providing evidence for its usefulness as a better screening test in cattle for the diagnosis of bovine genital campylobacteriosis.

Outbreak of caprine abortion by Toxoplasma gondii in Midwest Brazil

An outbreak of abortion by Toxoplasma gondii in goats on a farm in the Brazilian Midwest is reported. Gross lesions were not observed in seven aborted fetuses submitted to the Veterinary Pathology Laboratory, Federal University of Mato Grosso, for necropsy investigation. The main histologic lesions were mononuclear cell pneumonia and necrotizing encephalitis in varying degrees of intensity. PCR for Brucella abortus and Neospora caninum and aerobic cultures were negative in all cases. Antibody titles against T. gondii varying from 1:1024 to 1:32.768 were detected in serum samples from four aborted goats. Nested-PCR assay for T. gondii were positive in brain samples of all cases submitted. These findings indicate that T. gondii infection should be considered in the diagnosis of abortion in goats in Midwest Brazil.

Bronchoalveolar lavage as a tool for evaluation of cellular alteration during Aelurostrongylus abstrusus infection in cats

Bronchoalveolar lavage (BAL) is a procedure that retrieves cells and other elements from the lungs for evaluation, which helps in the diagnosis of pulmonary diseases. The aim of this study was to perform this procedure for cellular analysis of BAL fluid alterations during experimental infection with Aelurostrongylus abstrusus in cats. Fourteen cats were individually inoculated with 800 third stage larvae of A. abstrusus and five non-infected cats lined as a control group. The BAL procedure was performed through the use of an endotracheal tube on the nineteen cats with a mean age of 18 months, on 0, 30, 60, 90, 120, 180 and 270 days after infection. Absolute cell counts in the infected cats revealed that alveolar macrophages and eosinophils were the predominant cells following infection. This study shows that the technique allows us to retrieve cells and first stage larvae what provides information about the inflammatory process caused by aelurostrongylosis.

Kinematic gait analyses in healthy Golden Retrievers

Kinematic analysis relates to the relative movement between rigid bodies and finds application in gait analysis and other body movements, interpretation of their data when there is change, determines the choice of treatment to be instituted. The objective of this study was to standardize the march of Dog Golden Retriever Healthy to assist in the diagnosis and treatment of musculoskeletal disorders. We used a kinematic analysis system to analyse the gait of seven dogs Golden Retriever, female, aged between 2 and 4 years, weighing 21.5 to 28 kg, clinically normal. Flexion and extension were described for shoulder, elbow, carpal, hip, femorotibialis and tarsal joints. The gait was characterized lateral and had accepted hypothesis of normality for all variables, except for the stance of hip and elbow, considering a confidence level of 95%, significance level α = 0.05. Variations have been attributed to displacement of the stripes during movement and the duplicated number of reviews. The kinematic analysis proved to be a consistent method of evaluation of the movement during canine gait and the data can be used in the diagnosis and evaluation of canine gait in comparison to other studies and treatment of dogs with musculoskeletal disorders.

Social Networking Sites as a part of Medical Device Manufacturer's Marketing Communications

The purpose of the thesis is to examine how a medical device manufacturer can exploit social networking sites as a part of its everyday marketing communications. The ultimate goal is to create an ideal process of developing marketing communications in social networking sites as a medical device manufacturer with the help of theoretical knowledge and hands-on experience. Theoretical part examines the traditional process of developing marketing communications, defines social networking sites and presents marketing activities carried out on these sites as well as introduces the characteristics of healthcare technology industry. Empirical part is collected through participation in medical device manufacturer’s marketing operations and by observing effects of different factors and actions on social media marketing. In addition, completed interviews and a meeting with company’s personnel have been utilized for data collection. This part offers comprehensive information on the examined company’s current marketing operations, industry, and activities carried out on social networking sites. As a result of the thesis a comprehensive process description of integrating and using social networking sites as a part of company’s marketing communications was formed. With the help of the process description factors and actions which have an effect on marketing operations in social networking sites are presented and methods for further developing these activities are introduced.

NIR-Vis Up-Conversion Luminescence in the Yb3+,Er3+ Doped Y2O2S, ZrO2, and NaYF4 Nanomaterials

Since the discovery of the up-conversion phenomenon, there has been an ever increasing interest in up-converting phosphors in which the absorption of two or more low energy photons is followed by emission of a higher energy photon. Most up-conversion luminescence materials operate by using a combination of a trivalent rare earth (lanthanide) sensitizer (e.g. Yb or Er) and an activator (e.g. Er, Ho, Tm or Pr) ion in a crystal lattice. Up-converting phosphors have a variety of potential applications as lasers and displays as well as inks for security printing (e.g. bank notes and bonds). One of the most sophisticated applications of lanthanide up-conversion luminescence is probably in medical diagnostics. However, there are some major problems in the use of photoluminescence based on the direct UV excitation in immunoassays. Human blood absorbs strongly UV radiation as well as the emission of the phosphor in the visible. A promising way to overcome the problems arising from the blood absorption is to use a long wavelength excitation and benefit from the up-conversion luminescence. Since there is practically no absorption by the whole-blood in the near IR region, it has no capability for up-conversion in the excitation wavelength region of the conventional up-converting phosphor based on the Yb3+ (sensitizer) and Er3+ (activator) combination. The aim of this work was to prepare nanocrystalline materials with high red (and green) up-conversion luminescence efficiency for use in quantitative whole-blood immunoassays. For coupling to biological compounds, nanometer-sized (crystallite size below 50 nm) up-converting phosphor particles are required. The nanocrystalline ZrO2:Yb3+,Er3+, Y2O2S:Yb3+,Er3+, NaYF4:Yb3+,Er3+ and NaRF4-NaR’F4 (R: Y, Yb, Er) materials, prepared with the combustion, sol-gel, flux, co-precipitation and solvothermal synthesis, were studied using the thermal analysis, FT-IR spectroscopy, transmission electron microscopy, EDX spectroscopy, XANES/EXAFS measurements, absorption spectroscopy, X-ray powder diffraction, as well as up-conversion and thermoluminescence spectroscopies. The effect of the impurities of the phosphors, crystallite size, as well as the crystal structure on the up-conversion luminescence intensity was analyzed. Finally, a new phenomenon, persistent up-conversion luminescence was introduced and discussed. For efficient use in bioassays, more work is needed to yield nanomaterials with smaller and more uniform crystallite sizes. Surface modifications need to be studied to improve the dispersion in water. On the other hand, further work must be carried out to optimize the persistent up-conversion luminescence of the nanomaterials to allow for their use as efficient immunoassay nanomaterials combining the advantages of both up-conversion and persistent luminescence.

Biochemical profile of amniotic fluid for the assessment of fetal and renal development

Creatinine plays a key role in the function and maturation of fetal kidneys throughout pregnancy. It is important to identify other markers that may help in the diagnosis of renal dysfunction. Our aim was to determine the profile of and the correlation between biochemical markers to be used to assess renal function and maturation of the fetus in the amniotic fluid during pregnancy and to determine the distribution of normal values for creatinine, N-acetyl-ß-D-glucosaminidase (NAG), ß2-microglobulin, glucose, urea, sodium, potassium, phosphorus, calcium, uric acid, albumin, and osmolality in three gestational age groups. This was a cross-section study that assessed 115 samples of amniotic fluid during three different periods of pregnancy, i.e., 13 to 20, 27 to 34, and 36 to 42 weeks. Concentrations of creatinine, NAG, urea, potassium and uric acid increased during pregnancy (P<0.05). ß2-Microglobulin, glucose, sodium, phosphorus, calcium, and albumin concentration and osmolality decreased (P<0.05), whereas ß2-microglobulin, glucose and uric acid presented significant correlations with gestational age and creatinine, respectively (r>0.6, P<0.05). Urea, potassium and phosphorus showed mild correlations with both (r>0.5, P<0.05). NAG, sodium, albumin and osmolality did not show significant correlations (r<0.5, P<0.05). These tests confirmed the important role of creatinine in terms of correlation with gestational age. ß2-Microglobulin, glucose and uric acid were significant as markers of function and maturation of fetal kidneys, whereas NAG did not demonstrate a useful role for the assessment of renal maturation.

Nanoparticle-Assisted Immunoassays for Point-of-Care Testing - With Specific Interest in Minimally Interference-Prone Assays for Cardiac Troponin I

Cardiac troponins (cTn) I and T are the current golden standard biochemical markers in the diagnosis and risk stratification of patients with suspected acute coronary syndrome. During the past few years, novel assays capable of detecting cTn‐concentrations in >50% of apparently healthy individuals have become readily available. With the emerging of these high sensitivity cTn assays, reductions in the assay specificity have caused elevations in the measured cTn levels that do not correlate with the clinical picture of the patient. The increased assay sensitivity may reveal that various analytical interference mechanisms exist. This doctoral thesis focused on developing nanoparticle‐assisted immunometric assays that could possibly be applied to an automated point‐of‐care system. The main objective was to develop minimally interference‐prone assays for cTnI by employing recombinant antibody fragments. Fast 5‐ and 15‐minute assays for cTnI and D‐dimer, a degradation product of fibrin, based on intrinsically fluorescent nanoparticles were introduced, thus highlighting the versatility of nanoparticles as universally applicable labels. The utilization of antibody fragments in different versions of the developed cTnI‐assay enabled decreases in the used antibody amounts without sacrificing assay sensitivity. In addition, the utilization of recombinant antibody fragments was shown to significantly decrease the measured cTnI concentrations in an apparently healthy population, as well as in samples containing known amounts of potentially interfering factors: triglycerides, bilirubin, rheumatoid factors, or human anti‐mouse antibodies. When determining the specificity of four commercially available antibodies for cTnI, two out of the four cross‐reacted with skeletal troponin I, but caused crossreactivity issues in patient samples only when paired together. In conclusion, the results of this thesis emphasize the importance of careful antibody selection when developing cTnI assays. The results with different recombinant antibody fragments suggest that the utilization of antibody fragments should strongly be encouraged in the immunoassay field, especially with analytes such as cTnI that require highly sensitive assay approaches.

Leishmania infantum heat shock protein 83 for the serodiagnosis of tegumentary leishmaniasis

The serologic assay is an important tool in the diagnosis of leishmaniasis. One of the most commonly used tests is enzyme-linked immunosorbent assay (ELISA). Since total Leishmania promastigotes are used as antigen in the routine assay, false-positive reactions are frequent due to cross-reaction with sera from other diseases, mainly Chagas' disease. Therefore, an antigen that determines less cross-reactivity has been pursued for the serodiagnosis of leishmaniasis. In the present study we analyzed the use of recombinant Leishmania infantum heat shock protein (Hsp) 83 in ELISA for the serodiagnosis of cutaneous (N = 12) and mucocutaneous leishmaniasis (N = 14) and we observed the presence of anti-L. infantum Hsp 83 antibodies in all samples as well as anti-Leishmania total antigen antibodies. When cross-reactivity was tested, chronic Chagas' disease patients (N = 10) did not show any reactivity. Therefore, we consider this L. infantum Hsp 83 to be a good antigen for routine use for serodiagnosis of tegumentary leishmaniasis.

Automated grading of left ventricular segmental wall motion by an artificial neural network using color kinesis images

The present study describes an auxiliary tool in the diagnosis of left ventricular (LV) segmental wall motion (WM) abnormalities based on color-coded echocardiographic WM images. An artificial neural network (ANN) was developed and validated for grading LV segmental WM using data from color kinesis (CK) images, a technique developed to display the timing and magnitude of global and regional WM in real time. We evaluated 21 normal subjects and 20 patients with LVWM abnormalities revealed by two-dimensional echocardiography. CK images were obtained in two sets of viewing planes. A method was developed to analyze CK images, providing quantitation of fractional area change in each of the 16 LV segments. Two experienced observers analyzed LVWM from two-dimensional images and scored them as: 1) normal, 2) mild hypokinesia, 3) moderate hypokinesia, 4) severe hypokinesia, 5) akinesia, and 6) dyskinesia. Based on expert analysis of 10 normal subjects and 10 patients, we trained a multilayer perceptron ANN using a back-propagation algorithm to provide automated grading of LVWM, and this ANN was then tested in the remaining subjects. Excellent concordance between expert and ANN analysis was shown by ROC curve analysis, with measured area under the curve of 0.975. An excellent correlation was also obtained for global LV segmental WM index by expert and ANN analysis (R² = 0.99). In conclusion, ANN showed high accuracy for automated semi-quantitative grading of WM based on CK images. This technique can be an important aid, improving diagnostic accuracy and reducing inter-observer variability in scoring segmental LVWM.