Medizinische Universität Graz Austria/Österreich - Forschungsportal - Medical University of Graz
Gewählte Publikation:
Loibner, M.
Pre-analytic procedures for biomarker analysis
Doktoratsstudium der Medizinischen Wissenschaft; Humanmedizin; [ Dissertation ] Graz Medical University; 2018. pp.
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- Autor*innen der Med Uni Graz:
- Loibner Martina
- Betreuer*innen:
- Berghold Andrea
- Höfler Gerald
- Zatloukal Kurt
- Altmetrics:
- Abstract:
- Pre-analytic processes to which biological samples are subjected influence the outcome of all analytic methods. Sample taking, transport, storage conditions, physiological factors of the donors (age, sex, life style, etc.), and sample preparation comprise variables that not only impede reproducible results for biomarkers but also provoke tremendous costs in the health care system. Standards for molecular in vitro diagnostic examinations for snap frozen and formalin-fixed (FF) tissue, and DNA, RNA and protein isolation are available recently. Formalin crosslinks nucleic acids and proteins which requires harsh chemical conditions to isolate nucleic acids which hence become degraded. This negatively impacts the outcome of subsequent analyses. Highly sensitive molecular methods require high sample quality especially when sample size is limited. In addition during sample taking health care workers (HCWs) have to be protected from possible infections by adequate personal protective equipment (PPE). This work covers pre-analytic aspects from sample taking, biosafety issues and pathogen detection and ends at a selected example in routine diagnostic analysis over four chapters. Chapter 1: Sample taking from potentially high risk group infected or deceased patients became mandatory since an updated version of the Styrian Contagion Plan has been published in 2016. PPE protects HCWs but shall allow the same laboratory procedures without massive restrictions. A study with volunteers has been designed to test PPE systems by performing different tasks of simulated laboratory work. Contrary to expectations error rates did not increase and performances improved with ongoing study duration, although physical strain was measured. The results obtained were used to select the adequate PPE for the staff working in the newly built high security laboratory at the Institute of Pathology. Chapter 2: To improve sample quality, PAXgene Tissue, a non-crosslinking fixative was developed by the industrial partner of the Christian Doppler Laboratory for Biospecimen Research and Biobanking Technologies (Institute of Pathology). PAXgene, already extensively tested in an EU-project, preserves tissue morphology similar to FF tissue but the quality of isolated nucleic acids is superior. Hence, the question arose whether inactivation of bacterial, fungal and virus strains by PAXgene is as efficient as it is supposed to be with formalin. Except for one spore forming strain PAXgene inactivated all pathogens as well as formalin, which did also not achieve a sufficient inactivation. These results indicate that human samples may remain infectious after fixation. The same biosafety measures used for formalin can be applied for PAXgene. Chapter 3: The proven good quality of nucleic acids isolated from PAXgene-fixed (PF) samples raised the question of whether the sensitivity of virus detection may be improved when compared to FF material. No publications on this question as well as on the pathogen inactivation properties of PAXgene are available so far. The comparison of virus detection with two PCR methods showed a significantly higher sensitivity for PF compared to FF samples. Chapter 4: The determination of the HER2 (human epidermal growth factor receptor) status of breast cancer patients by FISH (fluorescence in situ hybridization) with an IVD-approved kit was used to investigate whether it can be applied on PF samples. The treatment of patients with herceptin is indicated when the HER2 gene locus is amplified. PF samples did not yield interpretable results before a pre-analytic treatment with formalin was performed for at least 16 hours. RNA quality tested from the same sample confirmed the feasibility of performing multiple analyses from a single PF sample with at least similar results for FISH and superior RNA quality when compared to FF material. Small samples indivisible for different analysis methods indicate the use of PAXgene as an alternative to formalin.