Verifikacija metoda za određivanje antitijela na acetilkolinske receptore (anti-AChR) i antitijela na mišićno-specifičnu tirozin-kinazu (anti-MuSK)

master's thesis

• Title (english): Verification of methods for the determination of antibodies to acetylcholine receptors (anti-AChR) and antibodies to muscle-specific tyrosine kinase (anti-MuSK)
• Author: Klaudija Žanetić
• Mentor: Donatella Verbanac (mentor)
• Mentor: Andrea Tešija Kuna (mentor)
• Committee member: Donatella Verbanac (predsjednik povjerenstva)
• Committee member: Andrea Tešija Kuna (član povjerenstva)
• Committee member: Lidija Bach-Rojecky (član povjerenstva)
• Granter: University of Zagreb; Faculty of Pharmacy and Biochemistry; (Department of medical biochemistry and haematology); Zagreb
• Defense date and country: 2023-07-06, Croatia
• Scientific / art field, discipline and subdiscipline: BIOMEDICINE AND HEALTHCARE; Pharmacy; Medical Biochemistry

Abstract

Miastenija gravis autoimuna je bolest koja najčešće nastaje zbog pojavnosti autoantitijela na acetilkolinski
receptor (anti-AchR) i autoantitijela na mišićno-specifičnu tirozin-kinazu (anti-MuSK) što uzrokuje
poremećeni neuromuskularni prijenos zbog čega nastaje mišićna slabost i umor. Cilj ovog rada je bio provesti
postupak verifikacije za dvije nove metode prije uvođenja u rutinski rad u Kliničkom zavodu za kemiju, KBC
Sestre milosrdnice. Ispitivane metode su kvantitativna ELISA metoda za određivanje anti-AChR i
kvalitativna metoda indirektne imunofluorescencije (IIF) na transfeciranim stanicama za određivanje antiMuSK. Nepreciznost ELISA metode za anti-AChR je ispitana mjerenjem komercijalnih kontrolnih uzoraka.
Rezultati su pokazali da ne zadovoljava kriterije deklarirane od proizvođača, ali zadovoljava kriterij kliničke
interpretacije kao i općeprihvaćeni kriterij nepreciznosti ELISA metoda. Istinitost ELISA metode za antiAChR je ispitana usporedbom s istovjetnom metodom implementiranom u Kliničkom zavodu za
laboratorijsku dijagnostiku, KBC Zagreb, na temelju 41 uzorka seruma. Ustanovljena je izvrsna podudarnost
u kategorizaciji rezultata između metoda. Regresijskom analizom ustanovljeno da u području klinički
značajnih pozitivnih rezultata ne postoji statistički značajno odstupanje između metoda. Ponovljivost IIF
metode za detekciju anti-MuSK antitijela je ispitana na 20 rezultata ponavljanja negativnih i pozitivnih
kontrolnih uzoraka te je zadovoljila postavljene kriterije prihvatljivosti prema CLSI EP12-A2. Istinitost IIF
metode je ispitana usporedbom s ELISA metodom implementiranom u Kliničkom zavodu za laboratorijsku
dijagnostiku, KBC Zagreb, na temelju 33 uzorka seruma. Ustanovljena je zadovoljavajuća usporedivost, ali
zbog malog broja pozitivnih uzoraka (mala pojavnost anti-MuSK) nije zadovoljen preduvjet za pravilnu
interpretaciju statističkog testa. Rezultati provedene verifikacije potvrđuju da su ispitivane metode
prihvatljive za uvođenje u rutinski rad.

Abstract (english)

Myasthenia gravis is an autoimmune disease that most often occurs due to the appearance of autoantibodies
to acetylcholine receptor (anti-AChR) and muscle-specific tyosine kinase (anti-MuSK), which causes
impaired neuromuscular transmission resulting in muscle weakness and fatigue. The aim of this study was to
conduct a verification procedure for two new methods before their implementation into routine work in the
Department of Clinical Chemistry, Sestre milosrdnice University Hospital. Tested methods were the
quantitative ELISA method for the determination of anti-AChR antibodies and the qualitative method of
indirect immunofluorescence (IIF) on transfected cells for the determination of anti-MuSK antibodies. The
imprecision of the ELISA method for anti-AChR was tested by measuring control samples. The imprecision
did not meet the criteria declared by the manufacturer, but meets the one of clinical interpretation as well as
the generally accepted criterion of imprecision of ELISA methods. The trueness of the ELISA method for
anti-AChR was tested by comparison with the identical method implemented in the Department of Laboratory
Diagnostics, University Hospital Centre Zagreb, based on 41 serum samples. A perfect agreement in the
categorization of the results between the methods was established and regression analysis revealed that there
is no statistically significant difference between the methods in the range of clinically significant results. The
reproducibility of the IIF method for the detection of anti-MuSK antibodies was tested on 20 repetition results
of negative and positive control samples and met the acceptance criteria according to CLSI EP12-A2. The
trueness of the IIF was tested by comparison with the ELISA method implemented in the Department of
Laboratory Diagnostics, University Hospital Centre Zagreb, based on 33 serum samples. A satisfactory
comparability was established, but due to the small number of positive samples (low incidence of anti-MuSK
antibodies), the precondition for the correct interpretation of the statistical test was not met. The results of the
verification confirm that the tested methods are acceptable for implementation into routine work.

• Keywords: anti-AChR; anti-MuSK; ELISA; indirektna imunofluorescencija; verifikacija
• Keywords (english): anti-AChR; anti-MuSK; ELISA; indirect immunofluorescence; verification
• Language: croatian
• URN:NBN: urn:nbn:hr:163:072586
• Study programme: Title: Medical biochemistry; Study programme type: university; Study level: integrated undergraduate and graduate; Academic / professional title: magistar/magistra medicinske biokemije (magistar/magistra medicinske biokemije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2023-11-21 14:52:54