Uspostava postupka za određivanje stupnja hemolize

master's thesis

• Title (english): Establishing a procedure for determining the degree of hemolysis
• Author: Nikolina Benc
• Mentor: Karmela Barišić (mentor)
• Mentor: Donatella Verbanac (mentor)
• Committee member: Karmela Barišić (predsjednik povjerenstva)
• Committee member: Donatella Verbanac (član povjerenstva)
• Committee member: Monika Barbarić (član povjerenstva)
• Granter: University of Zagreb; Faculty of Pharmacy and Biochemistry; (Department of medical biochemistry and haematology); Zagreb
• Defense date and country: 2021-09-07, Croatia
• Scientific / art field, discipline and subdiscipline: BIOMEDICINE AND HEALTHCARE; Pharmacy; Pharmacy

Abstract

In vivo (intravaskularna) hemoliza je relativno rijetko (1:10 000/100 000), ali životno ugrožavajuće stanje koje, između ostalog, može biti uzrokovano parenteralnom primjenom lijekova. Potencijalne komplikacije uključuju žuticu, hepatosplenomegaliju, tahikardiju, ishemiju miokarda, zatajenje bubrega i respiratorno zatajenje te u konačnici, multiorgansko zatajenje i smrt. Smrtnost može biti visoka, čak do 10 %. Zbog toga je važna procjena hemolitičkog potencijala lijekova tijekom pretkliničke faze ispitivanja lijekova. Cilj ovog rada bio je uspostaviti jednostavnu metodu za procjenu stupnja hemolize. Uzorci krvi tretirani su saponinom, tritonom-X-100 i propilen glikolom kako bi se izazvala hemoliza. Koncentracija hemoglobina određena je metodom s Drabkinovim reagensom te mjerena na dva tipa analizatora: spektrofotometru s kivetama (Cecil-CE 1011) i automatskom čitaču mikrotitarskih pločica (PerkinElmer-VICTOR3 1420 Multilabel Counter). Postotak hemolize izračunat je dijeljenjem apsorbancije uzoraka tretirane krvi s apsorbancijom uzoraka pune krvi. Metoda je praktična i lako primjenjiva, ali nije pogodna za analizu velikog broja uzoraka jer je dugotrajna i zahtjeva precizno pipetiranje. To se djelomično može poboljšati primjenom automatskog čitača mikrotitarskih pločica koji omogućava istovremenu analizu više uzoraka. Još jedan nedostatak je toksičnost Drabkinovog reagensa i štetnost za okoliš. Prednost metode je jeftin reagens i postojanje internacionalnog standarda.

Abstract (english)

In vivo (intravascular) hemolysis is relatively rare (1:10 000/100 000) but a life-threatening condition that, among other things, can be caused by parenteral administration of drugs. Potential complications include jaundice, hepatosplenomegaly, tachycardia, myocardial ischemia, renal failure and respiratory failure, and ultimately, multiorgan failure and death. Mortality can be as high as 10 %. Therefore, it is important to assess the hemolytic potential of drugs during the preclinical phase of drug testing. The aim of this study was to establish a simple method for estimating the percentage of hemolysis. Blood samples were treated with saponin, triton-X-100 and propylene glycol to induce hemolysis. The hemoglobin concentrations were determined by the Drabkin reagent method on two types of analyzers: a spectrophotometer with cuwette (Cecil-CE 1011) and an automatic microtiter plate reader (PerkinElmer-VICTOR3 1420 multilabel Counter). The percentage of hemolysis was calculated by dividing the absorbance of treated blood samples with the absorbance of whole blood samples. The method is practical and easy to apply, but it is not suitable for analysis of a large number of samples because it is time consuming and requires precise pipetting. This can be partially improved by using automatic microtiter plate reader that allows simultaneous analysis of multiple samples. Another disadvantage is toxicity of the Drabkin reagent and harmfulness to the environment. The advantage of the method is cheap reagent and existence of the international standard.

• Keywords: hemoliza; formulacije lijekova; ekscipijenti; ispitivanja lijekova
• Keywords (english): hemolysis; drug formulations; excipients; drug trials
• Language: croatian
• URN:NBN: urn:nbn:hr:163:340366
• 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
• Created on: 2022-02-04 12:31:08