Title Usporedba metoda mikrouzorkovanja krvi za analizu N-glikanskog profila plazme metodom HILIC-UPLC
Title (english) Blood microsampling method comparison for the HILIC-UPLC analysis of plasma N-glycan profile
Author Marin Ferić
Mentor Olga Gornik Kljaić (mentor)
Committee member Olga Gornik Kljaić (predsjednik povjerenstva)
Committee member Toma Keser (član povjerenstva)
Committee member Ana Mornar Turk (član povjerenstva)
Granter University of Zagreb Faculty of Pharmacy and Biochemistry (Department of biochemistry and molecular biology) Zagreb
Defense date and country 2021-09-16, Croatia
Scientific / art field, discipline and subdiscipline BIOMEDICINE AND HEALTHCARE Pharmacy Medical Biochemistry
Abstract Glikozilacija proteina najraznovrsnija je posttranslacijska modifikacija proteina, a N-glikozilacija najčešća je i najbolje proučena vrsta glikozilacije. Jasna je korelacija između N-glikanskog profila proteina plazme i brojnih patoloških stanja, naročito onih sa značajnom upalnom komponentom, što je analizu glikanskog profila proteina plazme učinilo vrlo perspektivnom. Poznavanje glikanskog profila plazme pojedinog pacijenta i njegova analiza visokoprotočnim metodama, kao što je HILIC-UPLC, uvelike bi doprinijelo personalizaciji medicinske intervencije. HILIC-UPLC metoda, osim što je visokoprotočna, vrlo je osjetljiva zbog čega je analiza glikanskog profila plazme dobivene venepunkcijom, koja je nepraktična i neugodna, zamijenjena analizom kapilarne krvi na filter papiru. Osim filter papira, u novije vrijeme na tržištu su postale dostupne brojne sofisticiranije metode za analizu kapilarne krvi.
U ovom radu ispitivane su performanse metoda za uzorkovanje kapilarne krvi na filter papiru (Whatman), Mitra štapiću (Neoteryx) i plazma separatoru (Noviplex) u svrhu analize glikanskog profila plazme. U prvoj fazi istraživanja analizirana je usporedivost navedenih metoda s uzorkom plazme dobivene venepunkcijom. U drugoj fazi istraživana je ponovljivost triju metoda za uzorkovanje kapilarne krvi.
U istraživanju usporedivosti korišteni su uzorci deset zdravih donora od kojih je prikupljen po jedan uzorak kapilarne krvi za tri ispitivane metode te po jedan uzorak venske krvi. Za analizu ponovljivosti korišteni su uzorci pet donora od kojih su prikupljeni uzorci triju metoda uzorkovanja kapilarne krvi u heksaplikatu. HILIC-UPLC metodom analizirani su N-glikanski profili proteina plazme, a dobiveni rezultati prikazani su metodama deskriptivne statistike.
Rezultati usporedivosti metoda za mikrouzorkovanje sa standardnim uzorkom plazme dobivene venepunkcijom pokazuju izvrsno slaganje rezultata uzoraka dobivenih plazma separatorom te vrlo dobro slaganje rezultata uzoraka dobivenih pomoću Mitra štapića. Spomenute metode mikrouzorkovanja pokazale su superiornost u odnosu na uzorak krvi na filter papiru.
Rezultati ponovljivosti triju metoda za mikrouzorkovanje krvi pokazuju najveću ponovljivost metode za uzorke dobivene plazma separatorom, a najslabiju ponovljivost metode na filter papiru.
Proučene performanse metoda za mikrouzorkovanje jasno ukazuju na analitičku superiornost metoda uzorkovanja plazma separatorom i Mitra štapićem u odnosu na filter papir, međutim ostale karakteristike metoda kao što su cijena, lakoća uzorkovanja, udio pravilno skupljenih uzoraka i mogućnost automatizacije također utječu na izbor metode uzorkovanja za analizu N-glikanskog profila plazme.
Abstract (english) Protein glycosylation is the most diverse posttranslational modification of proteins, with N-glycosylation being the most common one and the most extensively studied. The correlation between the N-glycan profile of plasma proteins and numerous pathological conditions is well known, particularly for those conditions with a significant inflammatory component. This notion made the analysis of the glycan profile of plasma proteins promising. Understanding the patient’s glycan profile and the ability to analyse it with high-throughput methods would contribute to the personalization of medical intervention considerably. HILIC-UPLC, besides being high-throughput, is a very sensitive analytical method allowing the replacement of impractical and unpleasant venepuncture sampling with a sampling of capillary blood on a filter paper. Along with the filter paper, numerous sophisticated methods for microsampling of capillary blood have recently been developed.
In this research filter paper (Whatman), Mitra sticks (Neoteryx) and plasma separator (Noviplex) devices for capillary blood microsampling were evaluated for the purpose of the plasma protein glycan profile analysis. The comparability of the mentioned microsampling methods with venepuncture were tested in the first phase of the research, whereas the repeatability of microsampling methods were assessed in the second phase.
In the first phase samples from ten healthy donors were acquired. One sample was collected for each microsampling method and venepuncture. In the second phase, the samples from five donors were collected in sextuplicate for each microsampling method. The analysis of the N-glycan profile of plasma proteins was done by the HILIC-UPLC. The collected data was interpreted by descriptive statistics.
Results of the first phase, i.e., comparability, showed the surpassing equivalence of the samples acquired by the plasma separator and a very good equivalence of the samples acquired by Mitra sticks to the standard venepuncture sampling. These microsampling methods have proved superior to the sampling on filter paper.
Results of the second phase, i.e., repeatability, attested the excellent repeatability of the plasma separator method and the lowest for sampling on the filter paper.
The studied performances of microsampling methods have clearly shown the analytical superiority of the plasma separator and Mitra stick sampling methods compared to the filter paper. However, other method properties, e.g., price of sampling devices, ease of sampling, degree of correctly sampled blood, and the ability of analysis automatization also take part in the selection of the sampling method for N-glycan profile analysis.
Keywords
N-glikani
HILIC
UPLC
mikrouzorkovanje
samouzorkovanje
DPS
VAMS
DBS
Keywords (english)
N-glycans
HILIC
UPLC
microsampling
self-sampling
DPS
VAMS
DBS
Language croatian
URN:NBN urn:nbn:hr:163:781396
Study programme Title: Medical biochemistry Study programme type: university Study level: integrated undergraduate and graduate Academic / professional title: magistar/magistra medicinske biokemije i laboratorijske medicine (magistar/magistra medicinske biokemije i laboratorijske medicine)
Type of resource Text
File origin Born digital
Access conditions Open access
Terms of use
Created on 2022-02-09 10:20:20