The influence of various sample storage conditions and sample microbial contamination on concentrations of routine biochemical parameters

Abstract

Background: The pre-analytical (PA) phase is the most vulnerable phase of laboratory testing procedure, with critical procedures-collection, handling, sample transport, and time and temperature of sample storage. The aim of this study was to examine if different anticoagulants, storage conditions, and freeze-thaw cycles (FTCs) influence the concentrations of basic biochemical parameters. In parallel, the presence and the effect of sample microbiological contamination during routine laboratory work were examined.

Methods: Two plasma pools (EDTA, and sodium-fluoride/potassium oxalate plasma (NaF)) were stored at +4C˚/-20˚C. Total cholesterol (TC), glucose, triglycerides (TG), urea, total protein (TP), and albumin concentrations were measured using Ilab 300+. Sample microbiological contamination was determined by 16S rRNA sequence analysis. The experiment encompassed a 5 day-period: Day 1–fresh sample, Day 2–1st FTC, Day 3–2nd FTC, Day 4–3rd FTC, Day 5–4th FTC. The appearance of bacteria in two consecutive samples was the experiment's endpoint.

Results: During 4 FTCs there were no changes in plasma urea concentrations. Glucose was stable in EDTA+4˚C and NaF- 20˚C until the 3rd FTC (P=0.008, P=0.042, respectively). Changes in protein concentrations followed the zig-zag pattern. TG concentrations changed significantly in the EDTA-20˚C sample after 1st and 4th FTCs (P=0.022, P=0.010, respectively). In NaF samples no contamination was observed during 4 FTCs.

Conclusions: Urea and glucose concentrations were robust. Changes in lipid and protein concentrations after FTCs follow complex patterns. Bacterial growth was not observed in NaF plasma samples. This can promote NaF use in analytical procedures in which microbiological contamination affects the quality of analysis.

Uvod: Preanaliti~ka (PA) faza je slo`en proces koji ~ine: prikupljanje, rukovanje, transport i skladi{tenje uzoraka, i predstavlja najzna~ajniji izvor laboratorijskih gre{aka. Cilj ovog istra`ivanja je bio da se ispita stabilnost osnovnih biohemijskih parametara u zavisnosti od uslova skladi{tenja uzoraka i broja ciklusa zamrzavanja-odmrzavanja (FTC). Pored toga, ispitivano je prisustvo bakterijske kontaminacije uzoraka tokom rutinskog laboratorijskog rada. Metode: Dva »pool«-a plazme (etilendiaminotetrasir}etna kiselina (EDTA) i natrijum-fluorid/kalijum oksalat (NaF)) su skladi{tena na +4 ˚C/-20 ˚C. Koncentracije ukupnog holesterola (TC), glukoze, triglicerida (TG), uree i albumina su odre|ene kori{}enjem BioSystems reagenasa (holesterol oksidaza/peroksidaza, glukoza oksidaza/peroksidaza, glice rol fosfat oksidaza/peroksidaza, ureaza/salicilat, od- nosno bromkrezol zeleno metodama, sukcesivno) na Ilab 300+ analizatoru. Bakterijska kontaminacija uzoraka je potvr|ena 16S rRNA sekvencioniranjem. Eksperiment je sproveden tokom 5 uzastopnih dana: 1. dan – sve` uzorak, 2. dan – 1. FTC, 3. dan –2. FTC, 4. dan – 3. FTC, 5. dan – 4. FTC. Zavr{nu ta~ku eksperimenta predstavljala je pojava bakterija u dva uzastopna uzorka. Rezultati: Tokom 4 FTC koncentracije uree u plazmi se nisu zna~ajno razlikovale. Koncentracija glukoze je bila stabilna u EDTA +4 ˚C i NaF -20 ˚C do 3.FTC (P=0,008, P=0,042, redom). Koncentracije TG su se zna~ajno pro- menile u uzorku EDTA -20 ˚C nakon 1. i 4. FTC-a (P=0,022, P=0,010, redom). U uzorcima NaF plazme nije do{lo do bakterijske kontaminacije tokom 4. FTC. Zaklju~ak: Koncentracije uree i glukoze su bile stabilne tokom trajanja eksperimenta. Promene u koncentracijama lipida nakon FTC prate slo`ene obrasce. Rast bakterija nije prime}en u uzorcima NaF plazme, te upotreba ovog anti- koagulansa mo`e biti opravdana u analiti~kim proce - durama podlo`nim uticaju mikrobiolo{ke kontaminacije.