Optimizacija in verifikacija metod prvega in drugega nivoja presejalnega testiranja novorojencev za cistično fibrozo iz vzorcev posušenega krvnega madeža

Založnik, Neža (author), Trebušak Podkrajšek, Katarina (mentor), Repič Lampret, Barbka (comentor)

Cistična fibroza je avtosomno-recesivna dedna bolezen, ki se najpogosteje pojavi pri kavkazijski populaciji in se izrazi pri približno 1:3500 živorojenih otrok. Bolezen se razvije pri otrocih, ki nosijo dve patološki genetski spremembi v področju gena CFTR na kromosomu 7 in prizadene številne organe kot so pljuča, trebušna slinavka, prebavila, jetra in reproduktivni sistem, posledica česar je krajša življenjska doba bolnikov. Namen magistrske naloge je bil vpeljati in verificirati metodi, ki se bosta uporabljali v okviru nacionalnega programa presejanja novorojencev v Sloveniji za cistično fibrozo na prvem (merjenje imunoreaktivnega tripsinogena – IRT) in drugem nivoju (merjenje s pankreatitisom povezanega proteina – PAP). V ta namen smo po modelu 5 x 5 za metodi DELFIA® Neonatal IRT in MucoPAP-F določili natančnost (znotraj in med serijami) in pravilnost. Pri določanju natančnosti metode DELFIA® Neonatal IRT smo izračunali koeficiente variacij za 3 kontrolne vzorce, ki so se znotraj serije gibali med 4,6 % in 5,8 %, med serijami pa med 1,9 % in 5,0 %. Pravilnost smo ocenili z odstopanjem povprečja meritev od tarčne vrednosti, kjer je bila pristranskost vseh treh kontrol med 1,1 % in 2,2 %. Nato smo po istem postopku ocenili še natančnost in pravilnost za metodo MucoPAP-F. Koeficienti variacij kontrolnih vzorcev znotraj serije so se gibali med 14,2 % in 19,2 %, med serijami med 3,4 % in 10,8 %, pristranskost pa med 1 % in 12 %. V zadnjem koraku smo določili še mejne vrednosti za IRT in v ta namen analizirali 2793 vzorcev novorojencev. Za mejo, ki bo predstavljala kriterij za uvrstitev pacienta v drugi nivo presejanja, smo izbrali 99. centil, kar ustreza 61,4 ng/ml. Pri 99,9. centilu (114,4 ng/ml) smo določili dodatno mejo za zelo visoke vrednosti, pri kateri se bodo pacienti uvrstili neposredno na potrditveno diagnostiko. Izmed vseh novorojencev, ki smo jih analizirali, so bili trije, ki so ustrezali temu kriteriju. Pri enem iz med njih so v laboratoriju kasneje odkrili patološko homozigotno spremembo gena CFTR in s tem potrdili cistično fibrozo. Z rezultati smo dokazali, da metodi dosegata ustrezne analitične kriterije in sta primerni za klinično uporabo. Presejalno testiranje novorojenev za cistično fibrozo v Sloveniji bo omogočilo prepoznavanje bolezni pri otrocih še pred pojavom kliničnih znakov in s tem takojšne zdravljenje ter posledično bolj kakovostno in daljše življenje bolnikov.

• Language: Slovenian
• Keywords: cistična fibroza, presejalno testiranje novorojencev, imunoreaktivni tripsinogen, s pankreatitisom povezan protein, verifikacija metode
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2024
• PID: 20.500.12556/RUL-162888
• Publication date in RUL: 28.09.2024

Secondary language

• Language: English
• Title: Optimisation and verification of first and second-tier methods for cystic fibrosis screening from neonatal dried blood spot samples

Abstract

Cystic fibrosis is an autosomal recessive genetic disorder that most commonly occurs in the Caucasian population, with an incidence of approximately 1 in 3,500 live births. The disease develops in children who carry two pathological genetic variant in the CFTR gene on chromosome 7 and affects multiple organs, including the lungs, pancreas, gastrointestinal tract, liver, and reproductive system, resulting in a shorter life expectancy for patients. The aim of this master's thesis was to implement and verify methods to be used in the national neonatal screening program for cystic fibrosis in Slovenia as the first (measuring immunoreactive trypsinogen – IRT) and the second-tier method (measuring pancreatitis-associated protein – PAP). To achieve this, we determined the precision (within and between series) and trueness of the DELFIA® Neonatal IRT and mucoPAP-F methods using a 5 x 5 model. For the DELFIA® Neonatal IRT method, we calculated the coefficients of variation for three control samples, which ranged from 4.6 % to 5.8 % within series and from 1.9 % to 5.0 % between series. Trueness was assessed by the bias of the average measurements against the target value, with biases ranging from 1.1 % to 2.2 % for all three controls. We then evaluated the precision and trueness of the mucoPAP-F method using the same procedure. The coefficients of variation for control samples within series ranged from 14.2 % to 19.2 %, and from 3.4 % to 10.8 % between series, while biases ranged from 1% to 12%. In the final step, we determined the cut-off values for IRT by analyzing 2793 neonatal samples. The 99th percentile, corresponding to 61.4 ng/ml, was selected as the cut-off value for referring a patient to the second-tier screening. An additional cut-off at the 99.9th percentile (114.4 ng/ml) was set for very high values, where patients would be directly referred for confirmatory diagnostics. Among all the newborns analyzed, three had IRT levels above this value. In one of these cases, a pathological homozygous CFTR variant was subsequently detected, confirming cystic fibrosis. The results demonstrated that both methods meet the appropriate analytical criteria and are suitable for clinical use. Neonatal screening for cystic fibrosis in Slovenia will enable early detection of the disease in children before the onset of clinical symptoms, allowing for immediate treatment and consequently a better quality of life and longer life expectancy for patients.

• Keywords: cystic fibrosis, newborn screening, immunoreactive trypsinogen, pancreatitis-associated protein, method verification