Vpliv hematokrita na določitev koncentracije ustekinumaba v posušenih krvnih madežih

Cirar, Matjaž (author), Vovk, Tomaž (mentor)

V terapiji Crohnove bolezni, ki je ena od oblik kronične vnetne črevesne bolezni, se za zdravljenje uporabljajo tudi biološka zdravila. Eno od njih je ustekinumab, človeško monoklonsko protitelo usmerjeno proti podenoti p40 prisotni v interlevkinu 12 in 23. Za nadzorovanje zdravljenja in napoved učinkovitosti terapije se v zadnjem času razvijajo pristopi, ki vključujejo terapevtsko spremljanje koncentracij. Pri teh pristopih je ponavadi potreben odvzem venske krvi v zdravstveni ustanovi, zato lahko pogosto vzorčenje pri bolnikih vzbuja nelagodje. V izogib temu lahko uporabljamo metode mikrovzorčenja, ki omogočajo samovzorčenje bolnikov v domačem okolju. Ena izmed takšnih metod je metoda posušenih krvnih madežev (DBS). Metoda je relativno enostavna, a je za točno in natančno kvantifikacijo analita potrebno premagati kar nekaj ovir. Eno izmed njih predstavlja vpliv hematokrita na volumen krvi v izseku posušenega krvnega madeža. V okviru magistrske naloge smo raziskovali ta vpliv in poskušali z upoštevanjem ugotovljenega odnosa izboljšati točnost metode za določevanje koncentracije ustekinumaba v vzorcih DBS. Za določitev volumna krvi smo razvili in prilagodili metodo na osnovi merjenja prevodnosti ekstraktov vzorcev DBS. Ugotovili smo, da je variabilnost volumna krvi zaradi hematokrita v izseku precejšnja in večja pri nanosu madežev večjih volumnov (8,4 – 12,0 µL pri 40 µL in 8,7 – 10,6 µL pri 20 µL madežih). V nadaljevanju smo skušali razviti hitre in enostavne metode za določitev vrednosti hematokrita v vzorcih DBS. Posredno smo hematokrit določevali z metodo določitve hemoglobina z uporabo reagenta z natrijevim lavrilsulfatom ter z metodo slikovne analize s programsko opremo ImageJ. Točnost rezultatov smo primerjali z rezultati referenčne metode (hematološki analizator). Za točnejšo in ponovljivejšo se je izkazala metoda slikovne analize. Pridobljeno znanje smo nato uporabili pri analizi realnih bolnikovih vzorcev. Koncentracijo ustekinumaba v vzorcih DBS smo določevali z encimsko imunsko metodo na trdnem nosilcu (ELISA). Metodo smo najprej poskušali prilagoditi in delno validirati za uporabo na vzorcih DBS, saj je bil uporabljeni komplet razvit in validiran le za uporabo na plazemskih ali serumskih vzorcih. Razvili smo metodo, sposobno določitve ustekinumaba v celotnem pričakovanem območju terapevtskih plazemskih koncentracij (3 – 120 mg/L). Ocenili smo točnost, natančnost metode in ustreznost umeritvene krivulje. V DBS določeno koncentracijo ustekinumaba smo nato na več načinov preračunali v plazemsko koncentracijo in jo primerjali s pravimi plazemskimi koncentracijami. Ugotovili smo, da z neupoštevanjem vrednosti hematokrita pri preračunu koncentracij vzorcev DBS pomembno napako storimo predvsem pri bolnikih, katerih vrednost hematokrita pomembneje odstopa od povprečja.

• Language: Slovenian
• Keywords: hematokrit, ustekinumab, posušeni krvni madeži, terapevtsko spremljanje koncentracij, slikovna analiza
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2020
• PID: 20.500.12556/RUL-121381
• Publication date in RUL: 07.10.2020

Secondary language

• Language: English
• Title: The haematocrit influence on ustekinumab concentration determination in dried blood spots

Abstract

In the therapy of Crohn's disease, which is a type of chronic inflammatory bowel disease, biological medicines are also used in the treatment. One of such is ustekinumab, a human monoclonal antibody targeted against the p40 subunit, which is present in interleukins 12 and 23. New approaches, using therapeutic drug monitoring as a method to monitor treatment with ustekinumab and to predict it's efficacy, are being developed. These approaches usually require regular sampling of patients' venous blood in health facilities, which can cause discomfort to the patients. We can overcome this problem by using microsampling methods, which allow home sampling. One of the methods is the dried blood spot method (DBS). The method is relatively simple, although we must overcome some challenges for precise and accurate quantification of the analyte. One of these challenges is the haematocrit influence on the blood volume present in the dried blood sample punch. We have tried to improve the precision of the method for the determination of ustekinumab by addressing this difficulty and estimating the relation between haematocrit value and blood volume in the sample. To measure the blood volume we developed and adapted a method based on conductivity measurement. We have found out that the volume variation in the punches due to haematocrit is significant and higher when a higher volume of blood is applied (8,4 – 12,0 µL at 40 µL and 8,7 – 10,6 µL at 20 µL spots). Additionally, we tried to develop quick and simple methods for the determination of haematocrit in DBS samples. We determined haematocrit indirectly by measuring haemoglobin using sodium lauryl sulfate reagent and by image analysis with ImageJ software. We compared the precision of both methods to the reference method (hematology analyzer). The image analysis method was found to be more precise and accurate. We have used the acquired knowledge in the analysis of real patient samples. To measure ustekinumab concentration, we used an enzyme-linked immunosorbent assay (ELISA) method. We tried to adapt and partially validate the method for use on DBS samples, as the kit we used, was developed and validated only for use with serum or plasma samples. We developed a method capable of determination of ustekinumab in the entire expected therapeutic plasma concentration range (3 – 120 mg/L). We investigated the precision and accuracy of the method and established the calibration curve. We converted the concentrations in DBS samples to plasma concentrations by using different methods and compared them to the real plasma concentrations. We have found out that by ignoring the haematocrit value in calculations, a significant error can be made especially with patients, whose haematocrit values significantly differ from average haematocrit values.

• Keywords: haematocrit, ustekinumab, dried blood spots, therapeutic drug monitoring, image analysis