Določanje krvne skupine Duffy z metodo verižne reakcije s polimerazo v realnem času v primerih imunohematološko neopredeljivih rezultatov za antigen Fy(b)

Turkalj, Iris (author), Jeras, Matjaž (mentor), Dovč Drnovšek, Tadeja (comentor)

V transfuzijski medicini je določitev antigenov krvnoskupinskega sistema Duffy pri krvodajalcih pomembna, saj morebitna prisotnost protiteles proti tem antigenom pri bolnikih povzroča nastanek hemolitične transfuzijske reakcije po transfuzijah v tej krvni skupini neskladnih eritrocitov. Na Zavodu RS za transfuzijsko medicino so pri določanju antigenov Fyb sistema Duffy krvodajalcev z uporabo novega avtomatiziranega sistema Erytra na gelskih karticah (Grifols) naleteli na neopredeljive rezultate. V okviru magistrskega dela smo zato želeli nadgraditi že delno vpeljano metodo qPCR za molekularno biološko določitev krvne skupine Duffy in pripraviti algoritem za testiranje krvodajalcev s serološko neopredeljivimi rezultati za antigen Fyb. Z optimizacijo reakcije qPCR za določitev prisotnosti polimorfizma posameznega nukleotida FY 265C>A z metodo alelske diskriminacije in s podatki iz literature, smo pri preiskovancih lahko pravilno napovedali prisotnost alelov FY*02W.01/.02, ki sta odgovorna za nastanek antigena Fyb+w. Na osnovi podatkov iz literature smo na genu FY izbrali polimorfno mesto -67T>C, optimizirali reakcijo qPCR in na podlagi naših rezultatov z metodo alelske diskriminacije qPCR lahko pravilno napovedali prisotnost alela FY*02N.01, ki je odgovoren za odsotnost antigena Fyb na eritrocitih. Reakcije qPCR smo izvedli na vzorcih krvodajalcev z neopredeljivimi serološkimi rezultati za antigen Fyb, pridobljenimi z novim sistemom Erytra. Pri vseh vzorcih smo s kombinacijo rezultatov genotipizacije uspeli določiti genotip in predvideti fenotip sistema Duffy. Pri tem, se je izkazalo, da je imelo le 50 % preiskovancev genotip FY*A/FY*02W.01/.02, torej fenotip Fy(a+b+w). Za pripravo algoritma testiranja krvodajalcev s serološko neopredeljivimi rezultati za antigen Fyb pri testiranju z analitskim sistemom Erytra, smo izvedli analizo dodatnih seroloških testov, opravljenih na gelskih karticah Bio-Rad in v epruvetah. Primerjava teh rezultatov z rezultati metode qPCR z alelsko diskriminacijo kot zlatim standardom ter z uporabo t.i. matrike zmede v velikosti šestih celic je pokazala, da je test v epruveti boljši serološki test. Rezultat našega dela je predlog algoritma, po katerem bi vzorce z neopredeljivimi rezultati za antigen Fyb na aparatu Erytra, v nadaljevanju testirali v epruveti. V primeru nadaljnjih negativnih in neopredeljivih rezultatov bi testiranje nadaljevali z metodo qPCR. Predlagani algoritem se je izkazal kot primeren za določanje prisotnosti antigena Fyb+w.

• Language: Slovenian
• Keywords: krvna skupina Duffy, serologija, genotipizacija, PCR v realnem času, algoritem testiranja.
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2023
• PID: 20.500.12556/RUL-151748
• Publication date in RUL: 19.10.2023

Secondary language

• Language: English
• Title: Blood group Duffy real-time polymerase chain reaction determination in cases of immunohematologically undeterminable results for the Fy(b) antigen

Abstract

Determination of Duffy blood group antigens in blood donors is important in transfusion medicine, since in patients with possible presence of antibodies against these antigens a hemolytic transfusion reaction after incompatible red blood cells transfusion can be developed. In Blood Transfusion Centre of Slovenia when the blood donors Fyb antigen of the Duffy blood group system is determining using the new automated Erytra system on gel cards (Grifols), the indeterminable results were encountered. The goal of the Master's thesis was to upgrade the partially implemented qPCR method for molecular determination of Duffy blood group and prepare an algorithm for testing blood donors with serologically indeterminable results for the Fyb antigen. After the optimization of the qPCR using the allelic discrimination approach for detection of FY 265C>A single nucleotide polymorphism and regarding the data from the literature, we were able to correctly predict the presence of the FY*02W.01/.02 alleles, responsible for the presence of Fyb+w antigen. Based on data from the literature, the FY -67T>C polymorphic site was selected, qPCR reaction was optimized and the presence of the FY*02N.01 allele, responsible for the absence of the Fyb antigen on erythrocytes, was correctly predicted using our qPCR results. qPCR reactions were performed on blood donor samples with indeterminable serological results for the Fyb antigen, using the new Erytra system. By combining the qPCR results we were able to to determine the genotype and predict the phenotype of the Duffy blood group system for all tested samples. Only 50 % have the genotype FY*A/FY*02W.01/.02 and therefore the phenotype Fy(a+b+w). In order to prepare an algorithm for testing blood donors with serologically indeterminable results obtained with the Erytra analytical system, an analysis of additional serological tests using Bio-Rad gel cards and test tubes were performed. A six-cell matrix was used to compare these results with qPCR results, which was determined as a gold standard, and the tube test was rated as a better serological test. The result of of our work was the proposal of an algorithm, according to which the samples with indeterminable results for the Fyb antigen, obtained during testing on the Erytra system, should be further tested in the tube. In case of negative and indeterminable results, the allelic discrimination qPCR testing could be performed. The proposed algorithm proved to be suitable for determining the presence of the Fyb+w antigen.

• Keywords: Duffy blood group, serology, genotyping, qPCR, algorithm for testing.