Preanalytical variables for liquid chromatography-mass spectrometry (LC-MS) analysis of human blood specimens.

The use of liquid chromatography-mass spectrometry (LC-MS) for both diagnostics and research purposes is rapidly growing in clinical laboratories. As for more conventional areas of in vitro diagnostic testing, many preanalytical variables have an impact on these techniques and may hence jeopardize the quality of tests results. The leading preanalytical variables include patient preparation, the nature of the blood collection tubes and additives, interference from spurious hemolysis, sample handling and management, composition of blood tubes, contamination, as well as storage conditions. Therefore, the aim of this article is provide a narrative overview about the leading preanalytical issues which may ultimately influence LC-MS testing of human blood samples, and provide tentative indications, as for current evidence, about optimal preanalytical management of blood samples for proteomics and metabolomics studies. These general recommendations entail pre-storage centrifugation, use of appropriate tubes and additives, addition of bacteriostatic preservatives, enrichment and purification of samples, elimination of unsuitable specimens, rapid analysis or immediate storage at -70°C, and avoidance of analyzing frozen-thawed specimens.

Salvagno GL ，Danese E ，Lippi G 《-》

Blood venous sample collection: Recommendations overview and a checklist to improve quality.

The extra-analytical phases of the total testing process have substantial impact on managed care, as well as an inherent high risk of vulnerability to errors which is often greater than that of the analytical phase. The collection of biological samples is a crucial preanalytical activity. Problems or errors occurring shortly before, or soon after, this preanalytical step may impair sample quality and characteristics, or else modify the final results of testing. The standardization of fasting requirements, rest, patient position and psychological state of the patient are therefore crucial for mitigating the impact of preanalytical variability. Moreover, the quality of materials used for collecting specimens, along with their compatibility, can guarantee sample quality and persistence of chemical and physical characteristics of the analytes over time, so safeguarding the reliability of testing. Appropriate techniques and sampling procedures are effective to prevent problems such as hemolysis, undue clotting in the blood tube, draw of insufficient sample volume and modification of analyte concentration. An accurate identification of both patient and blood samples is a key priority as for other healthcare activities. Good laboratory practice and appropriate training of operators, by specifically targeting collection of biological samples, blood in particular, may greatly improve this issue, thus lowering the risk of errors and their adverse clinical consequences. The implementation of a simple and rapid check-list, including verification of blood collection devices, patient preparation and sampling techniques, was found to be effective for enhancing sample quality and reducing some preanalytical errors associated with these procedures. The use of this tool, along with implementation of objective and standardized systems for detecting non-conformities related to unsuitable samples, can be helpful for standardizing preanalytical activities and improving the quality of laboratory diagnostics, ultimately helping to reaffirm a "preanalytical" culture founded on knowledge and real risk perception.

Giavarina D ，Lippi G 《-》

Preanalytical standardization for reactive oxygen species derived oxysterol analysis in human plasma by liquid chromatography-tandem mass spectrometry.

The analysis of the oxysterols 7-keto-, 7-α/β-hydroxy-, 5α,6α-epoxy-, 5β,6β-epoxycholesterol and cholestane-3β,5α,6β-triol derived from reactive oxygen species (ROS) is of interest as biomarkers in the field of atherosclerosis. Preanalytical validation is a crucial point to minimize the susceptibility of oxysterols to in vitro autoxidation. The aim of this study was to standardize a preanalytical protocol for ROS-derived oxysterol analysis by liquid chromatography-tandem mass spectrometry in human plasma. Sample matrices were compared and stability of free oxysterols in whole blood and EDTA-plasma was investigated with regard to short-term storage until sample preparation, freeze-thaw cycles, addition of butylated hydroxytoluene and long-term storage up to 1 year at different temperatures (-20 °C, -80 °C and -130 °C) as well as different storage containers (safe-lock tubes, cryo tubes and straws). Sample preparation prior LC-MS/MS analysis was reduced to a simple concentration and protein precipitation step. Storing EDTA-whole blood for 30 min at room temperature resulted in <25% concentration changes, within acceptable change limits (ACL). In freshly prepared plasma samples, free oxysterols were stable for 90 min stored at 4 °C with concentration changes <23.5% (within ACL). Up to nine freeze-thaw cycles did not affect analyte concentrations (concentration change -8.5% to +5.0%). 7-Ketocholesterol was stable for 2 years stored <-80 °C; concentration changes below 20.5% (within ACL). The remaining oxysterols were stored for a maximum of 2-4 weeks without exceeding ACL. The addition of BHT did not reveal improvement in analyte stability for storage at -80 or -130 °C. We developed a standardized preanalytical protocol for oxysterol analysis based on LC-MS/MS, compared cryobanking conditions for each oxysterol and present data for long-term storage up to 2 years.

Helmschrodt C ，Becker S ，Thiery J ，Ceglarek U ... -  《-》

Preanalytical Issues in Hemostasis and Thrombosis Testing.

Lippi G ，Favaloro EJ 《-》

Preanalytical aspects and sample quality assessment in metabolomics studies of human blood.

Metabolomics is a powerful tool that is increasingly used in clinical research. Although excellent sample quality is essential, it can easily be compromised by undetected preanalytical errors. We set out to identify critical preanalytical steps and biomarkers that reflect preanalytical inaccuracies. We systematically investigated the effects of preanalytical variables (blood collection tubes, hemolysis, temperature and time before further processing, and number of freeze-thaw cycles) on metabolomics studies of clinical blood and plasma samples using a nontargeted LC-MS approach. Serum and heparinate blood collection tubes led to chemical noise in the mass spectra. Distinct, significant changes of 64 features in the EDTA-plasma metabolome were detected when blood was exposed to room temperature for 2, 4, 8, and 24 h. The resulting pattern was characterized by increases in hypoxanthine and sphingosine 1-phosphate (800% and 380%, respectively, at 2 h). In contrast, the plasma metabolome was stable for up to 4 h when EDTA blood samples were immediately placed in iced water. Hemolysis also caused numerous changes in the metabolic profile. Unexpectedly, up to 4 freeze-thaw cycles only slightly changed the EDTA-plasma metabolome, but increased the individual variability. Nontargeted metabolomics investigations led to the following recommendations for the preanalytical phase: test the blood collection tubes, avoid hemolysis, place whole blood immediately in ice water, use EDTA plasma, and preferably use nonrefrozen biobank samples. To exclude outliers due to preanalytical errors, inspect the biomarker signal intensities reflecting systematic as well as accidental and preanalytical inaccuracies before processing the bioinformatics data.

Yin P ，Peter A ，Franken H ，Zhao X ，Neukamm SS ，Rosenbaum L ，Lucio M ，Zell A ，Häring HU ，Xu G ，Lehmann R ... -  《-》