Variability in, variability out: best practice ...

Clin Chem Lab Med 2017; 55(5): 608–621

Review Jenna Khana, Joshua A. Liebermana and Christina M. Lockwood*

Variability in, variability out: best practice recommendations to standardize pre-analytical variables in the detection of circulating and tissue microRNAs DOI 10.1515/cclm-2016-0471 Received June 1, 2016; accepted September 5, 2016; previously published online March 17, 2017

Abstract: microRNAs (miRNAs) hold promise as biomarkers for a variety of disease processes and for determining cell differentiation. These short RNA species are robust, survive harsh treatment and storage conditions and may be extracted from blood and tissue. Pre-analytical variables are critical confounders in the analysis of miRNAs: we elucidate these and identify best practices for minimizing sample variation in blood and tissue specimens. Preanalytical variables addressed include patient-intrinsic variation, time and temperature from sample collection to storage or processing, processing methods, contamination by cells and blood components, RNA extraction method, normalization, and storage time/conditions. For circulating miRNAs, hemolysis and blood cell contamination significantly affect profiles; samples should be processed within 2 h of collection; ethylene diamine tetraacetic acid (EDTA) is preferred while heparin should be avoided; samples should be “double spun” or filtered; room temperature or 4 °C storage for up to 24 h is preferred; miRNAs are stable for at least 1 year at –20 °C or –80 °C. For tissuebased analysis, warm ischemic time should be  7 indicates a high risk of hemolysis [28]. Although it would be ideal to completely exclude all hemolyzed specimens, this may not be feasible or practical. A modified workflow for hemolyzed specimens has been proposed that excludes hemolyzed specimens from initial biomarker discovery, but that then conditionally allows for their inclusion in the final analysis if the miRNAs of interest are not affected by hemolysis [5]. The evaluation of hemolysis alone, however, is inadequate to account for blood cell-derived changes in miRNA concentrations. A recent study demonstrated hemolysisindependent release of primarily vesicle-associated blood cell miRNAs miR-16 and miR-21. This release occurred during the first 5 h of room temperature storage of whole blood, prior to separation of plasma or serum from cellular components [52]. Notably, increased concentrations were absent for miRNAs not associated with blood cell expression, such as liver-specific miR-122. Thus, there is a need for meticulous specimen processing and storage documentation as well as evaluation of blood cell associated miRNAs in each specimen. Another underappreciated variable that can affect miRNA quantification is plasma volume. Plasma inherently contains polymerase inhibitors such as hemoglobin, lactoferrin, and IgG, which can co-purify with nucleic acids. A comprehensive analysis demonstrated that optimizing plasma volume is critical for accurate quantification: too little plasma will have insufficient miRNA for quantification and too much plasma can increase polymerase inhibitors. One study suggested 50 μL of serum or plasma optimized miRNA detection using SYBR Green or TaqMan qRT-PCR (11-fold and three-fold, respectively) compared to 200 and 10 μL [55]. Another study demonstrated better recovery with 200 μL of plasma compared to higher volumes of up to 500 μL [37]. Whether putative polymerase inhibitors interfere with miRNA detection depends on multiple factors including both extraction and quantification methods. Additional processing, such as enriching small RNAs and silica adsorption can be used to effectively remove inhibitors [55]. Depending on miRNA detection methods, a combination of polymerases resistant to blood-borne inhibitors could be an alternative workaround.

Specimen processing Blood sample processing requires centrifugation to remove cellular components. Although plasma is considered the cell-free fluid portion of blood, it typically contains residual platelets and microparticles. It is essential to recognize that even trace amounts of contaminating platelets or microparticles will artificially increase miRNAs, and potentially obscure disease-related miRNA expression profiles because cellular miRNA concentrations are so much higher than fluid concentrations. Centrifugation speed can significantly impact miRNA concentrations either by inadequately removing cellular components (slow spin) or by causing cell lysis (fast spin). The influence of platelets and microparticles on miRNAs in matched fresh plasma and serum was systematically evaluated by measuring miRNAs after two-step centrifugation (slower 1940 g with no brake for 10  min followed by faster 3400  g with high brake for 10 min) and 0.22 μm filtration [30]. Significant differences were identified in 72% of the measured miRNAs due to processing (4× to > 1000× variation in expression). Notably, the miRNAs most affected were those with the highest expression in platelets. Another study examined the effects of two-step centrifugation (1000 g followed by either 1000 g, 2000 g, or 10,000 g) and found that much faster spins of 10,000 g substantially reduced platelet-associated miRNAs [36]. A separate study showed that two-step centrifugation effectively removed platelets from frozen plasma and serum specimens stored for up to 6 years [30]. Incorporation of an additional centrifugation step during specimen processing is highly recommended to remove confounding platelet miRNAs [3, 5, 30]. Alternatively, filtration can remove platelets and cell debris. Although platelet-derived miRNAs accounted for the majority of differences observed, filtration additionally removed microparticle miRNAs [30]. The use of filtration should be considered based on the relevance of microparticle miRNAs to the study objectives. A post-processing platelet count with rejection of specimens above a predefined threshold can also be incorporated to further improve standardization [30]. In laboratory medicine, it is well established that delayed processing can cause significant changes in many measured analytes [13]. It is therefore not unexpected that delayed specimen processing affects miRNAs. For example, a delay in centrifugation of 6 h compared to 2 h caused significant variation in six miRNA profiles, particularly miR-15b and miR-191 [36]. An additional study investigating the effect of delayed processing showed an initial selective increase of vesicle-associated miRNAs at 1–3 h, followed by a steady decline in all miRNAs measured up to 24  h [52]. They also demonstrated that the Brought to you by | University of Washington Libraries Authenticated Download Date | 5/4/17 4:54 PM

614      Khan et al.: Pre-analytical variables in the detection of circulating and tissue microRNAs decrease in miRNA concentrations could be ameliorated by the addition of an RNaseA inhibitor. These studies illustrate the need for rapid specimen processing that may include freezing separated plasma/serum or using a stabilizing agent such as an RNase inhibitor to maximize miRNA yield.

Specimen storage Circulating miRNAs are typically preserved by storage at temperatures low enough to significantly reduce RNase activity rather than by chemical fixation. Fortunately, studies to date suggest that samples can be stored as processed/separated plasma/serum for at least 24  h either at room temperature or 4 °C [2, 33, 37, 47]. Furthermore, miRNAs are relatively stable and can be stored for at least 1  year at –20 °C or –70/80 °C [37]. A recent study compared multiple storage conditions on miR-134 and miR346 concentrations in whole blood specimens measured by qRT-PCR [57]. There was no significant change due to processing delay, storage conditions, or storage duration; however, increased freeze-thaw cycles significantly decreased miRNA concentrations. Another storage study at −80 °C and −20 °C showed good stability of miRNAs for up to 6 years [58]. Interestingly, they found overall similar amounts of miRNAs when comparing storage at −80 °C and −20 °C but differential expression of miRNAs depending on the storage temperature, suggesting that storage temperatures should be consistent throughout a study. A separate study demonstrated similar miRNA profiles and expression following storage at −80 °C for 12  years [36]. Since there is minimal and occasionally conflicting data about the effects of long-term storage (multiple years), control specimens matched for storage time should be considered [35]. Freeze-thaw cycles can potentially affect multiple analytes and conflicting results have been reported on the impact of freeze-thaw cycles on miRNAs [47, 57–59]. Thus, minimizing freeze-thaw cycles is recommended.

Pre-analytical variables for ­tissue-based analysis of miRNAs Human tissue, most often fixed in formalin and embedded in paraffin (FFPE), is the cornerstone of diagnostic pathology. Pathology laboratories archive clinical tissue samples as FFPE “blocks” and these stored specimens are an invaluable resource for basic, translational, and clinical

science. Although not routinely used for clinical diagnostic purposes, analysis of miRNAs in such specimens is increasingly common, with the hope that miRNA signatures will supplement morphology, along with protein expression, transcriptional profiles, and DNA sequence analysis as important adjuncts for clinicians and anatomic pathologists. Analysis in situ provides important advantages, including direct association of miRNA signatures with histologically confirmed disease process. Thus, analysis of miRNAs in tissue not only may discover signatures associated with specific disease states, particularly solid neoplasms, but is also a necessary step to validate miRNA species identified as diagnostic, predictive, or prognostic markers. Two themes related to pre-analytical variables emerge from review of the tissue miRNA literature. First, miRNAs are robust, and, when compared to other nucleic acids, better tolerate both direct and tissue insults. For example, various markers of total RNA integrity including electropherograms [55, 56, 59–61] and RNA integrity number (RIN) [57, 61] do not correlate with miRNA integrity, which consistently remains detectable despite overall specimen decay. Second, while standardized pre-analytical conditions for tissue samples are ideal, it is not always practical and variation in the above variables should be recorded and analyzed as potential confounders.

Ischemic time after sample procurement A notable variable in clinical tissue samples is time the tissue spends at either room temperature or at 4 °C following excision and the resulting loss of blood supply prior to freezing or fixing. The warm and cold ischemic time, respectively, vary significantly from sample to sample and both have profound and deleterious effects on downstream recovery of nucleic acids and protein antigens [62, 63]. In addition, hypoxic/ischemic injury is a major biological stress that can induce significant transcriptional changes; the degree to which hypoxia alters miRNA expression profiles is an open area of research. The degree of ischemic effects, particularly tissue autolysis, varies significantly with tissue type. For example, pancreatic tissue is particularly sensitive given the high concentration of intracellular peptidases [64]. Unfortunately, the ischemic time is not readily controllable for most clinical samples and is generally not routinely recorded. Few studies have systematically examined the effect of ischemic time on miRNA recovery, further confounding attempts to establish best practices. Brought to you by | University of Washington Libraries Authenticated Download Date | 5/4/17 4:54 PM

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A recent study carefully assessed the effects of ischemic time preceding fixation or freezing on miRNA expression of autopsy cardiac tissue following myocardial infarct (also known as the post mortem interval) [60]. In this study, the post mortem interval ranged from 10 h to 154 h; cadavers were stored at room temperature for 3 h–24 h prior to cold storage at 4 °C. High quality miRNAs were detectable from cardiac tissue up to a week following death and the concentration of miRNAs varied less than that of other small RNA species. Despite the ability of miRNAs to withstand harsh treatment, the authors suggest prompt transfer to 4 °C to better preserve miRNA integrity. They further note that electrophoretic profiling of total RNA likely underestimates the population of intact miRNA since longer RNA species are more readily degraded than shorter molecules, which can be visualized as a smear on gel electrophoresis. Multiple studies have documented changes in miRNA expression profiles following even short ischemic events. One study demonstrated time-dependent changes in the expression of 56 miRNAs and 1788 mRNAs during warm ischemic time [65]. The most prominent effects (particularly for miRNAs) occurred after 1 h of warm ischemic time; thus, the authors recommend freezing tissue at 30  min of warm ischemic time [65]. A more recent study using a gerbil model of cerebral ischemia examined the effects of 2  min carotid artery occlusion on miRNA expression in the hippocampus [66]. They identified seven miRNAs upregulated following transient carotid artery ligation and concluded that these changes are a neuroprotective mechanism against ischemic injury. Although miRNAs tend to remain stable after tissue is removed from a patient, ischemic time can nonetheless potentially change miRNA expression or, to a lesser degree, cause sample degradation. Confounding effects are that time dependent and warm ischemia are more potent than cold ischemia. Uncertainty about total ischemic time is of particular concern in retrospective analysis of archived tissue samples, whereas prospective studies may incorporate time and/or recording standards. In addition to recording and recognizing warm and cold ischemic times as sources of variability, we offer specific recommendations for prospective studies in Table  3. For studies of archived tissue, we recommend collecting ischemic time if available. If unavailable, a common miRNA control whose expression is not affected by ischemia is critical.

Fixative, fixation time and tissue storage For clinical samples, neutral buffered formalin is the fixative of choice for optimal evaluation by pathologists.

Table 3: Tissue-specific best practice recommendations. –– Ischemic time