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Optimizacija metode štetja mišjih retikulocitov za potrebe določanja in vivo biološke aktivnosti eritropoetina.
Kovač, Vito (Author), Marinšek Logar, Romana (Mentor) More about this mentor... This link opens in a new window, Škrajnar, Špela (Co-mentor)

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Abstract
Za zdravljenje anemije, ki je posledica ledvičnih motenj, se uporablja rekombinantni človeški eritropoetin (rHuEPO). Njegovo aktivnost smo testirali s standardiziranim in vivo biološkim testom na mišji liniji B6D2F1 z uporabo fluorescenčne pretočne citometrije, pri čemer smo kvantitativno ovrednotili povišanje deleža retikulocitov v krvi, ki se pojavi kot odziv na injiciran rHuEPO. Cilj magistrske naloge je bil optimizirati analitski test za obarvanje retikulocitov in določiti najboljši čas vzorčenja mišje krvi. Potrdili smo, da je najboljši čas vzorčenja četrti dan po injiciranju substance. Preučili smo tudi različne vplive na pogoje barvanja retikulocitov v krvnih vzorcih. Krvne vzorce smo pobarvali s komercialnim fluorescenčnim barvilom BD Retic-Count in laboratorijsko pripravljenim fluorescenčnim barvilom tiazol oranžno v različnih koncentracijah. Inkubacija vzorcev v barvilu je potekala pod različnimi pogoji (čas, svetloba, vorteksiranje). Z rezultati smo pokazali, da sta barvili primerljivi, vendar obstajajo razlike med obarvanostjo s komercialnim barvilom BD Retic-Count in laboratorijsko pripravljenim barvilom. Optimalna inkubacija vzorca v laboratorijsko pripravljenem barvilu je med 30 minut in 150 minut ne glede na prisotnost ali odsotnost svetlobe. Pred merjenjem na pretočnem citometru je priporočljivo enkratno kratkotrajno vorteksiranje vzorca zaradi zagotovitve homogenosti vzorca. Z boljšim poznavanjem analitskih metod in analitske opreme lahko zagotavljamo boljšo kontrolo kakovosti in s tem omogočamo, da je terapevtsko zdravilo varno in učinkovito za uporabo.

Language:Slovenian
Keywords:eritropoetin, retikulociti, pretočni citometer, tiazol oranžno
Work type:Master's thesis/paper (mb22)
Tipology:2.09 - Master's Thesis
Organization:BF - Biotechnical Faculty
Year:2018
Publisher:[V. Kovač]
UDC:602:57.088:606:62(043.2)
COBISS.SI-ID:9047161 Link is opened in a new window
Views:623
Downloads:273
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Secondary language

Language:English
Title:Optimization of mouse reticulocyte counting method for in vivo determination of erythropoietin biological activity
Abstract:
Recombinant human erythropoietin (rHuEPO) is an effective drug for curing people with anemia (low blood count) due to kidney dysfunction. To test its activity, we used a standardized in vivo bioassay protocol on mice line B6D2F1 with an automated fluorescence flow cytometry. The purpose of the master thesis was to optimize the analytical assay in reticulocyte staining and to determinate the best appropriate time for mouse blood sampling. We have confirmed that the optimal sampling is 4 days after drug application. We have quantitatively evaluated the increase of reticulocyte proportion in blood samples, which occurs as a response to the injected rHuEPO. Different influences for reticulocytes dying conditions were also studied. Blood samples were stained with commercial dye BD Retic-Count and laboratory prepared fluorescent dye thiazole orange in different concentrations and left incubated under different conditions (time, light, and vortex). The study showed that the staining process with commercial dye BD Retic-Count is comparable but different to laboratory prepared dye. The best incubation time of sample stained with laboratory prepared dye is between 30 minutes and 150 minutes regardless of the light presence. Before measuring on the flow cytometer, a onetime short vortexing is recommended to ensure homogeneity of the sample. With the better understanding of analytical methods and equipment, we can ensure better quality control which is the base for safe and effective therapeutic product.

Keywords:erythropoietin, reticulocytes, flow cytometer, thiazol orange

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