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  Validering af automatiseret haptoglobinassay til hund: med henblik på rutinemæssig anvendelse samt en vurdering af de kliniske anvendelsesmuligheder
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 Ophav:
Stephansen, Synne1, Forfatter
Kjelgaard-Hansen, Mads1, Vejleder
Tilknytninger:
1Det Biovidenskabelige Fakultet, København, Danmark
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 Abstract: Acute phase response (APR) is a part of the innate immune system in both humans and animals. APR is initiated by tissue damage and causes a range of metabolic, endocrinal and neurological changes. One of the central aspects in APR is the changes in the synthesis og acute phase proteins in the liver. The negative acute phase proteins' synthesis is inhibited. The positive acute phase proteins' synthesis is stimulated, and the concentration of these in the blood rises. Acute phase proteins are used as an indicator of inflammatory conditions. Acute phase proteins are closely related to inflammation, and their synthesis is influenced relatively quickly after the inflammatory stimulus; how quickly varies from protein to protein. At the Small Animal University Hospital at Faculty of Life Science, University of Copenhagen (KU-LIFE), the acute phase proteins C-reactive protein (CRP) and fibrinogen is routinely measured in blood samples from dogs. The object of this master thesis was to validate and implement an automated assay for routine measurement of the acute-phase protein haptoglobin (Hp) in dogs. The concentration of Hp in the blood of dogs rises and normalises slower than CRP, but normalises quicker than fibrinogen. By applying routine measurements of Hp in blood samples from dogs, the ability to date and monitor inflammatory conditions is optimised. As assay the spectrophotometric assay PhaseTM Range Hp Assay from Tridelta Development Limited (Tridelta's Hp Assay) was chosen. This choice was made from the desire for an automated assay, which could be set up on the analysis device Siemens ADVIA 1800, which is already in use at The Small Animal University Hospital at KU-LIFE. A fundamental requirement to a laboratorie is to have analysis results reflect the conditions of the patients. Hence it is important to uncover and minimise the errors present in analyses. This is done via evaluation and validation of new assays and analysis devices. The evaluation is concerned with the laboratorial applicability of the assay. Phase 1 of the validation is concerned with the analytical performance of the assay, while phase 2 and 3 deals with the clinical performance. The laboratorial applicability of Trideltas Hp Assay is inhibited by reagent II's limited durability. According to the study of reagent stability, reagent II requires a renewal of a freshly mixed substance every hour, which causes a great workload as well as a large volume of waste. The linearity study and the intra-assay precision of the analytical validation of Trideltas Hp Assay obtained the standard criterias. The precision of the inter-assay was not large enough. In the method comparison of Trideltas Hp Assay with an immunological assay, variations in the Hp measurements beyond the imprecision of the assays were seen. In the clinical validation of Trideltas Hp Assay, it was shown that the assay is capable of distinguishing between healthy dogs and dogs with pathological conditions with a 95% confidence interval. The further clinical validation was hypothetical and theoretical, as the patient material was too small and too selective for a proper fase II and III validation. An Hp-cut-off value between sick and healthy dogs was estimated to 1500 mg/L. 136 dogs were hereafter placed in one of four groups, depending on whether their Hp and CRP concentrations were above or below the respective cut-off. The theories behind the placement were held together with data from the dogs' jounals. The result of Tridelta's Hp Assay's validation was that the laboratorial applicability of the assay is not satisfactory due to the instability of the reagents. Furthermore the precision in the inter-assay was not evaluated as large enough. Tridelta's Hp Assay was therefore not implemented as a routine blood parameter on dog plasma at the Small Animal University Hospital at KU-LIFE.
 Abstract: Akutfaserespons (APR) er en del af det medfødte immunforvar hos mennesker og dyr. APR ses ved vævsskader og medfører en række metabolske, endokrine og neurologiske ændringer. Centralt er leverens ændring i syntesen af akutfaseproteiner. De negative akutfaseproteiners syntese hæmmes, mens de positive akutfaseproteiners syntese stimuleres og deres koncentration i blodet stiger. Akutfaseproteiner anvendes som markører for inflammatoriske tilstande. Akutfaseproteiner er specifikt relateret til inflammation og deres syntese påvirkes forholdsvist hurtigt efter det inflammatoriske stimulus; hvor hurtigt varierer fra protein til protein. På Hospital For Mindre Husdyr på Det Biovidenskabelige Fakultet, Københavns Universitet (KU-LIFE) måles akutfaseproteinerne C-reaktivt protein (CRP) og fibrinogen rutinemæssigt i blodprøver fra hunde. Målet med dette speciale er, at validere og implementere et automatiseret assay til rutinemæssig måling af akutfaseproteinet haptoglobin (Hp) hos hund. Hp´s koncentration i blodet hos hunde stiger og normaliseres langsommere end CRP, men normaliseres hurtigere end fibrinogen. Ved tilførelsen af rutinemæssige Hp-målinger hos hund optimeres klinikerens mulighed for tidsfæstelse og monitorering af inflammatoriske tilstande. Som assay valgtes det spektrofotometriske assay PhaseTM Range Hp Assay fra Tridelta Development Limited (Trideltas Hp Assay). Valget er gjort udfra ønsket om et automatiseret assay, der kan opsættes på analyseapparatet Siemens ADVIA 1800, som allerede anvendes på Hospital For Mindre Husdyrs på KU-LIFE. Den fornemste opgave for laboratorier er at få analyseresultaterne til at afspejle patienternes tilstand. Derfor er det vigtigt at synliggøre og minimere de afvigelser, der sker ved analyser. Dette gøres via evaluering og validering af nye assays og analyseapparater. Evalueringen beskæftiger sig med assayets laboratoriemæssige anvendelighedsgrad. Valideringens fase 1 vurderer assayets analytiske egenskaber mens fase 2 og 3 omhandler den kliniske anvendelighed. Den laboratoriemæssige anvendelighed for Trieltas Hp Assay hæmmes af reagens II´s begrænsede holdbarhed. Ifølge et reagensstabilitetsstudie kræver reagens II udskifting med friskblandet hver time, medførerende en stor arbejdsbyrde samt et stort spildvolumen. Ved den analytiske validering af Tridelta Hp Assay opfyldte linearitetsstudiet samt intra-assayets præcision de opstillede kriterier. Inter-assayets præcision var ikke stor nok. Ved metodesammenligning af Trideltas Hp Assay med et immunologisk assay, sås der variationer i Hp-målinger ud over de to assays samlede upræcision. Ved den kliniske validering af Trideltas Hp Assay var assayet med 95% sikkerhed er i stand til at skelne mellem raske hunde og hunde med patologiske tilstande. Den videre kliniske validering var hypotetisk og teoretisk, da patientmaterialet var for lille og for selekteret. En Hp-cut-off værdi mellem syge og raske hunde blev estimeret til 1500 mg/L. 136 hunde blev herefter placeret i en af fire grupper dannet på baggrund af om Hp- og CRP-koncentrationen lå over eller under det respektive cut-off. Teorier bag placeringen af patienter blev sammenholdt med hundenes journaldata. Resultatet af Trideltas Hp Assays validering blev, at assayets laboratoriemæssige anvendelighed ikke er tilfredsstillende grundet reagensinstabilitet. Herudover blev præcisionen ved inter-assay ikke vurderet stor nok. Trideltas Hp Assay er derfor ikke blevet implementeret til rutinekørsler på hundeserum på Hospital For Mindre Husdyrs på KU-LIFE.
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Bogmærk denne post: https://diskurs.kb.dk/item/diskurs:12255:1
 Type: Speciale
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Detaljer

Sprog: Danish - dan
 Datoer: 2009
 Sider: 74 pages
 Publiceringsinfo: København, Danmark : Institut for Mindre Husdyrs Sygdomme
 Indholdsfortegnelse: -
 Note: Veterinary Medicine, Veterinærmedicin
 Type: Speciale
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