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When dealing with pre-analytics for tissues, it is often that case that tissue heterogeneity, and particularly tumor heterogeneity, is not taken into account as a preliminary condition for obtaining reproducible results in molecular analysis at the diagnostics and clinical research levels. It is well known that when sampling tumor tissues in different areas, for example the border or the central area of the tumor, different genes are expressed, and due to polyclonality in most tumors different areas can have different DNA and epigenetic alterations. For this reason, it is extremely important to establish and standardize specific tissue sampling protocols for molecular extraction as well as in situ molecular methods. A correct approach to heterogeneity is the basis for a more reproducible and exchangeable type of molecular analysis that can provide useful information at the prognostic and predictive levels. Heterogeneity should also be taken into consideration during cancer treatment, since therapy modifies the clonal composition of tumors. Here the different types of tumor heterogeneity and the improper pre-analytical conditions in tissue processing that can generate heterogeneous artefacts are described.

BACKGROUND: The recommendation for coagulation blood samples is to centrifuge at 4000 revolutions per minute (rpm) for 15 min to produce platelet-poor plasma before analysis. Rapid centrifugation, defined as centrifuging samples at higher speeds for shorter durations, could potentially reduce turn-around times (TAT), provided sample integrity is maintained. OBJECTIVE: This study assessed the impact of rapid centrifugation on routine coagulation assay results. METHODS: Blood samples were collected from volunteers at Inkosi Albert Luthuli Central Hospital and King Edward VIII Hospital, Durban, KwaZulu-Natal, South Africa, from September to November 2021. Samples were centrifuged using Method A, the current standard (4000 rpm/15 min), Method B (4000 rpm/10 min), Method C (5000 rpm/10 min) and Method D (5000 rpm/5 min). Platelet count, prothrombin time, activated partial thromboplastin time, thrombin time (TT), fibrinogen and D-dimer levels were analysed and results from Methods B, C and D compared to reference Method A. RESULTS: Platelet-poor plasma was obtained from all samples (n = 60) using Methods A and B, and from 33/60 (55%) samples using Methods C and D. Differences between Method A and Methods C and D for normal prothrombin time, normal D-dimer and abnormal TT results were statistically significant. Prothrombin time results correlated strongly across all methods, while TT and D-dimer results correlated poorly. Activated partial thromboplastin time and fibrinogen results showed no significant differences across all methods. CONCLUSION: Rapid centrifugation at 4000 rpm/10 min (Method B) showed results consistent with the reference method. This method could potentially reduce the overall TAT for routine coagulation assays.

Background Hemoglobin (Hb) is a hemeprotein with two linked pairs of globin chains. Each chain is connected to a heme residue in its center. Hemoglobinopathies are divided into quantitative and qualitative defects in globin synthesis. Hemolysis is a pre-analytical problem that reduces quality of sample for measurement of many analytes. Methods Blood samples from 311 female and 189 male subjects with normal and abnormal Hb electrophoresis patterns were studied. Three milliliters of whole blood was obtained from all subjects and transferred into EDTA tubes. To analyze hemolysis, 1.5 ml of blood from each tube was aliquoted and frozen, and the remaining blood was stored at 4 °C for 24 h. Hemoglobin was measured in both hemolyzed and non-hemolyzed samples by capillary electrophoresis. Results and discussion Data was analyzed with linear regression. The results were linear for the lower limit of detection of 9, 0.5, and 0.1% up to at least 99.5, 6.6, and 99.4% for HbA, HbA2, and HbF, respectively. Method comparison demonstrated good agreement between non-hemolyzed and hemolyzed conditions for hemoglobin measurement. Conclusion Use of hemolyzed samples had no effected on hemoglobin measurements.

The stability of lipids and other metabolites in human body fluids ranges from very stable over several days to very unstable within minutes after sample collection. Since the high-resolution analytics of metabolomics and lipidomics approaches comprise all these compounds, the handling of body fluid samples, and thus the pre-analytical phase, is of utmost importance to obtain valid profiling data. This phase consists of two parts, sample collection in the hospital (“bedside”) and sample processing in the laboratory (“bench”). For sample quality, the apparently simple steps in the hospital are much more critical than the “bench” side handling, where (bio)analytical chemists focus on highly standardized processing for high-resolution analysis under well-controlled conditions. This review discusses the most critical pre-analytical steps for sample quality from patient preparation; collection of body fluids (blood, urine, cerebrospinal fluid) to sample handling, transport, and storage in freezers; and subsequent thawing using current literature, as well as own investigations and practical experiences in the hospital. Furthermore, it provides guidance for (bio)analytical chemists to detect and prevent potential pre-analytical pitfalls at the “bedside,” and how to assess the quality of already collected body fluid samples. A knowledge base is provided allowing one to decide whether or not the sample quality is acceptable for its intended use in distinct profiling approaches and to select the most suitable samples for high-resolution metabolomics and lipidomics investigations. Graphical abstract

Background: Urine contamination is a widely established clinical problem that can generate unreliable results leading to misdiagnosis and improper or delayed treatments. Diagnostic stewardship programs focus on the pre-analytic phase with an aim to improve patient outcomes by reducing contamination rates and improving specimen collection and processing. Methods: Utilizing Edward Deming’s Plan-Do-Study-Act model for quality improvement, a retrospective chart review was conducted to evaluate if an educational in-service, introducing a 3-step pre-analytic diagnostic stewardship protocol, would affect the rates of urine contamination. Data collected was analyzed by a pre- and post-implementation method using Chi-Square test for independence. Results: This project did not demonstrate a significant relationship between educational in-services on a pre-analytic protocol, and urinalysis contamination rates, (χ² (1, n = 1303) = .01, p = .93, phi = -.01). However, there was a significance association identified amongst culture contamination rates and implementation of such education sessions (χ² (1, n=791) = 3.78, p = 0.05, phi = -.07). Conclusion: This project demonstrates that education can reduce urine contamination rates. These results, and those previously published in the literature, suggest that education in addition to mandating a pre-analytic protocol can prove to be both statistically and clinically significant. Future projects that have the ability to evaluate such combined methods may deliver a larger clinical impact.

Ovarian cancer kills more than 40 000 women in Europe and more than 150 000 women globally each year. High-grade serous ovarian cancer (HGSOC) is the most common and most difficult to treat subtype of the disease. The high-grade serous tumors are highly heterogeneous, therefore, though most of the patients respond well to surgery and chemotherapy initially, more than half experience relapse. HERCULES is a research project funded by the EU H2020 program with the target to characterize comprehensively high grade serous ovarian cancers to find novel therapeutic strategies to fight them. The aim of my PhD thesis was to validate biomarkers identified within HERCULES project in a retrospective case study of patients affected by HGSOC, studying tumor heterogeneity and evaluating the effects of pre-analytical variables, in particular fixation, in the validation process. High grade serous ovarian cancer samples were characterized validating selected biomarkers at both RNA and protein level. Molecular analysis and in situ analysis were performed on multiple tissue biopsies in order to detect spatial heterogeneity and, moreover, biomechanical proprieties of fixed tumor tissues were measured. Lastly, the reliability of molecular analyses on archive tissues were assessed, determining the effect of formalin and Bouin’s fixation at RNA level and evaluating their impact on gene expression using different platforms. Our results showed that detail morphological and immunophenotypical analyses, at the level of the entire tissue slide and not of TMA spots (Tissue micro array), of HGSOC tumors are paramount for the differential diagnosis as well as for both prognostication and therapy. In this view, biomechanical properties, by AFM can support the morphological findings. Among the immunohistochemical markers, Ki67 and BRCA1 have been shown their predictive value for response to first line chemotherapy and overall survival in HGSOC patients. Furthermore, neo adjuvant chemotherapy seems to have a detrimental effect on patient in our cohort. At the RNA level, cyclin C and HLA-B biomarkers showed their prognostic value indicating longer overall survival, while AKTs isoforms have shown a different impact on patients’ outcome. Regarding the pre-analytical variables, fixation confirmed to have a deep impact on molecular analyses, especially in RNA expression. Tissues with highly fragmented RNA such those fixed in Bouin’s can lead to analytical bias in both ddPCR, RT-qPCR, Nanostring and RNAscope technologies. A careful selection of samples with proper nucleic acids quality and integrity is of paramount importance before starting any molecular analysis. Also in that case, to minimize the effect of sample to sample variability a proper sample size should be used. Bouin’s fixed samples because of their high level of nucleic acids fragmentation are not recommended for mRNA expression analyses, especially for low expressed targets. Contrarily, miRNAs, giving their length, are more resistant to fixation procedures and can be used for RNA expression analyses in both formalin and Bouin’s tissues after a proper method of normalization

In May 2017, the European In Vitro Diagnostic Regulation (IVDR) entered into force and will apply to in vitro diagnostics from May 26th, 2022. This will have a major impact on the in vitro diagnostics (IVD) industry as all devices falling under the scope of the IVDR will require new or re-certification. It will also affect health institutions developing and using in-house devices. The IVDR also has implications with respect to product performance validation and verification including the pre-analytics of biological samples used by IVD developers and diagnostic service providers. In parallel to the IVDR, a series of standards on pre-analytical sample processing has been published by the International Organization for Standardization (ISO) and the European Committee for Standardization (CEN). These standards describe pre-analytical requirements for various types of analyses in various types of biospecimens. They are of relevance for IVD product developers in the context of (re)certification under the IVDR and to some extent also to devices manufactured and used only within health institutions. This review highlights the background and the rational for the pre-analytical standards. It describes the procedure that leads to these standards, the major implications of the standards and the requirements on pre-analytical workflows. In addition, it discusses the relationship between the standards and the IVDR.

INTRODUCTION: We have recently described epitope detection in macrophages (EDIM) by flow cytometry. This is a promising tool for the diagnosis and follow-up of malignancies. However, biological and technical validation is warranted before clinical applicability can be explored. METHODS: The pre-analytic and analytic phases were investigated. Five different aspects were assessed: blood sample stability, intra-individual variability in healthy persons, intra-assay variation, inter-assay variation and assay transferability. The post-analytic phase was already partly standardized and described in an earlier study. RESULTS: The outcomes in the pre-analytic phase showed that samples are stable for 24h after venipuncture. Biological variation over time was similar to that of serum tumor marker assays; each patient has a baseline value. Intra-assay variation showed good reproducibility, while inter-assay variation showed reproducibility similar to that of to established serum tumor marker assays. Furthermore, the assay showed excellent transferability between analyzers. CONCLUSION: Under optimal analytic conditions the EDIM method is technically stable, reproducible and transferable. Biological variation over time needs further assessment in future work.