Standardisation and harmonisation of thyroid-stimulating hormone measurements: historical, current, and future perspectives.

Thyroid-stimulating hormone (TSH) is an important clinical marker in the diagnosis and management of thyroid disease. TSH measurements are reported in milli-International Units per Litre (mIU/L), traceable to a World Health Organisation (WHO) reference material. There is a wide variety of commercial immunoassays for TSH measurements available, which have historically been poorly harmonised due to a lack of commutability of the WHO reference materials with patient samples. This led to the recent development of a serum-based reference panel for TSH, traceable to the WHO reference material, available via the International Federation for Clinical Chemistry and Laboratory Medicine (IFCC), aimed at harmonisation of TSH immunoassays. This report describes recent developments in the TSH reference system, including establishment of the 4th WHO International Standard for TSH, and aims to clarify the relationship between the available reference materials and their intended uses. This 4th WHO IS is widely available and defines the unit of TSH activity, therefore its continued existence is of paramount importance, however it continues to show a lack of commutability with patient in many TSH immunoassays. This makes the C-STFT TSH panel, albeit available in restricted numbers, a critical resource to ensure better TSH assay harmonisation.

Development of an LC-MRM-MS-Based Candidate Reference Measurement Procedure for Standardization of Serum Apolipoprotein (a) Tests.

BACKGROUND: Medical results generated by European CE Marking for In Vitro Diagnostic or in-house tests should be traceable to higher order reference measurement systems (RMS), such as International Federation of Clinical Chemistry and Laboratory Medicine (IFCC)-endorsed reference measurement procedures (RMPs) and reference materials. Currently, serum apolipoprotein (a) [apo(a)] is recognized as a novel risk factor for cardiovascular risk assessment and patient management. The former RMS for serum apo(a) is no longer available; consequently, an International System of Units (SI)-traceable, ideally multiplexed, and sustainable RMS for apo(a) is needed. METHODS: A mass spectrometry (MS)-based candidate RMP (cRMP) for apo(a) was developed using quantitative bottom-up proteomics targeting 3 proteotypic peptides. The method was provisionally validated according to ISO 15193 using a single human serum based calibrator traceable to the former WHO-IFCC RMS. RESULTS: The quantitation of serum apo(a) was by design independent of its size polymorphism, was linear from 3.8 to 456 nmol/L, and had a lower limit of quantitation for apo(a) of 3.8 nmol/L using peptide LFLEPTQADIALLK. Interpeptide agreement showed Pearson Rs of 0.987 and 0.984 for peptides GISSTVTGR and TPENYPNAGLTR, and method comparison indicated good correspondence (slopes 0.977, 1.033, and 1.085 for LFLEPTQADIALLK, GISSTVTGR, and TPENYPNAGLTR). Average within-laboratory imprecision of the cRMP was 8.9%, 11.9%, and 12.8% for the 3 peptides. CONCLUSIONS: A robust, antibody-independent, MS-based cRMP was developed as higher order RMP and an essential part of the apo(a) traceability chain and future RMS. The cRMP fulfils predefined analytical performance specifications, making it a promising RMP candidate in an SI-traceable MS-based RMS for apo(a).

Commutability Assessment of Candidate Reference Materials for Lipoprotein(a) by Comparison of a MS-based Candidate Reference Measurement Procedure with Immunoassays.

BACKGROUND: Elevated concentrations of lipoprotein(a) [Lp(a)] are directly related to an increased risk of cardiovascular diseases, making it a relevant biomarker for clinical risk assessment. However, the lack of global standardization of current Lp(a) measurement procedures (MPs) leads to inconsistent patient care. The International Federation for Clinical Chemistry and Laboratory Medicine working group on quantitating apolipoproteins by mass spectrometry (MS) aims to develop a next-generation SI (International system of units)-traceable reference measurement system consisting of a MS-based, peptide-calibrated reference measurement procedure (RMP) and secondary serum-based reference materials (RMs) certified for their apolipoprotein(a) [apo(a)] content. To reach measurement standardization through this new measurement system, 2 essential requirements need to be fulfilled: a sufficient correlation among the MPs and appropriate commutability of future serum-based RMs. METHODS: The correlation among the candidate RMP (cRMP) and immunoassay-based MPs was assessed by measuring a panel of 39 clinical samples (CS). In addition, the commutability of 14 different candidate RMs was investigated. RESULTS: Results of the immunoassay-based MPs and the cRMPs demonstrated good linear correlations for the CS but some significant sample-specific differences were also observed. The results of the commutability study show that RMs based on unspiked human serum pools can be commutable with CS, whereas human pools spiked with recombinant apo(a) show different behavior compared to CS. CONCLUSIONS: The results of this study show that unspiked human serum pools are the preferred candidate secondary RMs in the future SI-traceable Lp(a) Reference Measurement System.

Recommendations for Setting a Criterion and Assessing Commutability of Sample Materials Used in External Quality Assessment/Proficiency Testing Schemes.

It is important for external quality assessment materials (EQAMs) to be commutable with clinical samples; i.e., they should behave like clinical samples when measured using end-user clinical laboratory in vitro diagnostic medical devices (IVD-MDs). Using commutable EQAMs makes it possible to evaluate metrological traceability and/or equivalence of results between IVD-MDs. The criterion for assessing commutability of an EQAM between 2 IVD-MDs is that its result should be within the prediction interval limits based on the statistical distribution of the clinical sample results from the 2 IVD-MDs being compared. The width of the prediction interval is, among other things, dependent on the analytical performance characteristics of the IVD-MDs. A presupposition for using this criterion is that the differences in nonselectivity between the 2 IVD-MDs being compared are acceptable. An acceptable difference in nonselectivity should be small relative to the analytical performance specifications used in the external quality assessment scheme. The acceptable difference in nonselectivity is used to modify the prediction interval criterion for commutability assessment. The present report provides recommendations on how to establish a criterion for acceptable commutability for EQAMS, establish the difference in nonselectivity that can be accepted between IVD-MDs, and perform a commutability assessment. The report also contains examples for performing a commutability assessment of EQAMs.

Recommendations for Setting a Criterion for Assessing Commutability of Secondary Calibrator Certified Reference Materials.

A secondary higher-order calibrator is required to be commutable with clinical samples to be suitable for use in the calibration hierarchy of an end-user clinical laboratory in vitro diagnostic medical device (IVD-MD). Commutability is a property of a reference material that means results for a reference material and for clinical samples have the same numeric relationship, within specified limits, across the measurement procedures for which the reference material is intended to be used. Procedures for assessing commutability have been described in the literature. This report provides recommendations for establishing a quantitative criterion to assess the commutability of a certified reference material (CRM). The criterion is the maximum allowable noncommutability bias (MANCB) that allows a CRM to be used as a calibrator in a calibration hierarchy for an IVD-MD without exceeding the maximum allowable combined standard uncertainty for a clinical sample result (umaxCS). Consequently, the MANCB is derived as a fraction of the umaxCS for the measurand. The suitability of an MANCB for practical use in a commutability assessment is determined by estimating the number of measurements of clinical samples and CRMs required based on the precision performance and nonselectivity for the measurand of the measurement procedures in the assessment. Guidance is also provided for evaluating indeterminate commutability conclusions and how to report results of a commutability assessment.

Assessment of WHO 07/202 reference material and human serum pools for commutability and for the potential to reduce variability among soluble transferrin receptor assays.

OBJECTIVES: The clinical use of soluble transferrin receptor (sTfR) as an iron status indicator is hindered by a lack of assay standardization and common reference ranges and decision thresholds. In 2009, the WHO and National Institute for Biological Standards and Controls (NIBSC) released a sTfR reference material (RM), 07/202, for assay standardization; however, a comprehensive, formal commutability study was not conducted. METHODS: This study evaluated the commutability of WHO 07/202 sTfR RM and human serum pools and the impacts of their use as common calibrators. Commutability was assessed for six different measurement procedures (MPs). Serum pools were prepared according to updated CLSI C37-A procedures (C37) or non-C37 procedures. The study design and analyses were based on Parts 2 and 3 of the 2018 IFCC Commutability in Metrological Traceability Working Group's Recommendations for Commutability Assessment. WHO 07/202 and serum pools were used for instrument/assay and mathematical recalibration, respectively, to determine if their use decreases inter-assay measurement variability for clinical samples. RESULTS: The WHO 07/202 RM dilutions were commutable for all 6 MPs assessed and, when used for instrument calibration, decreased inter-assay variability from 208 to 55.7 %. Non-C37 and C37 serum pools were commutable for all 6 MPs assessed and decreased inter-assay variability from 208 to 13.8 % and 4.6 %, respectively, when used for mathematical recalibration. CONCLUSIONS: All materials evaluated, when used as common calibrators, substantially decreased inter-assay sTfR measurement variability. MP calibration to non-C37 and C37 serum pools may reduce the sTfR IMPBR to a greater extent than WHO 07/202 RM.

Osteopontin in Vascular Disease.

Inflammatory cytokines are necessary for an acute response to injury and the progressive healing process. However, when this acute response does not resolve and becomes chronic, the same proteins that once promoted healing then contribute to chronic inflammatory pathologies, such as atherosclerosis. OPN (Osteopontin) is a secreted matricellular cytokine that signals through integrin and CD44 receptors, is highly upregulated in acute and chronic inflammatory settings, and has been implicated in physiological and pathophysiologic processes. Evidence from the literature suggests that OPN may fit within the Goldilocks paradigm with respect to cardiovascular disease, where acute increases are protective, attenuate vascular calcification, and promote postischemic neovascularization. In contrast, chronic increases in OPN are clinically associated with an increased risk for a major adverse cardiovascular event, and OPN expression is a strong predictor of cardiovascular disease independent of traditional risk factors. With the recent finding that humans express multiple OPN isoforms as the result of alternative splicing and that these isoforms have distinct biologic functions, future studies are required to determine what OPN isoform(s) are expressed in the setting of vascular disease and what role each of these isoforms plays in vascular disease progression. This review aims to discuss our current understanding of the role(s) of OPN in vascular disease pathologies using evidence from in vitro, animal, and clinical studies. Where possible, we discuss what is known about OPN isoform expression and our understanding of OPN isoform contributions to cardiovascular disease pathologies.

Osteopontin isoforms differentially promote arteriogenesis in response to ischemia via macrophage accumulation and survival.

Osteopontin (OPN) is critical for ischemia-induced neovascularization. Unlike rodents, humans express three OPN isoforms (a, b, and c); however, the roles of these isoforms in post-ischemic neovascularization and cell migration remain undefined. Our objective was to determine if OPN isoforms differentially affect post-ischemic neovascularization and to elucidate the mechanisms underlying these differences. To investigate if human OPN isoforms exert divergent effects on post-ischemic neovascularization, we utilized OPN-/- mice and a loss-of-function/gain-of-function approach in vivo and in vitro. In this study OPN-/- mice underwent hindlimb ischemia surgery and 1.5 × 106 lentivirus particles were administered intramuscularly to overexpress OPNa, OPNb, or OPNc. OPNa and OPNc significantly improved limb perfusion 30.4% ± 0.8 and 70.9% ± 6.3, respectively, and this translated to improved functional limb use, as measured by voluntary running wheel utilization. OPNa- and OPNc-treated animals exhibited significant increases in arteriogenesis, defined here as the remodeling of existing arterioles into larger conductance arteries. Macrophages play a prominent role in the arteriogenesis process and OPNa- and OPNc-treated animals showed significant increases in macrophage accumulation in vivo. In vitro, OPN isoforms did not affect macrophage polarization, whereas all three isoforms increased macrophage survival and decreased macrophage apoptosis. However, OPN isoforms exert differential effects on macrophage migration, where OPNa and OPNc significantly increased macrophage migration, with OPNc serving as the most potent isoform. In conclusion, human OPN isoforms exert divergent effects on neovascularization through differential effects on arteriogenesis and macrophage accumulation in vivo and on macrophage migration and survival, but not polarization, in vitro. Altogether, these data support that human OPN isoforms may represent novel therapeutic targets to improve neovascualrization and preserve tissue function in patients with obstructive artery diseases.

Impaired Collateral Vessel Formation in Sickle Cell Disease.

OBJECTIVE: The adaptive response to vascular injury is the formation of functional collateral vessels to maintain organ integrity. Many of the clinical complications associated with sickle cell disease can be attributed to repeated bouts of vascular insufficiency, yet the detailed mechanisms of collateral vessel formation after injury are largely unknown in sickle cell disease. Here, we characterize postischemic neovascularization in sickle cell disease and the role of neutrophils in the production of reactive oxygen species. APPROACH AND RESULTS: We induced hindlimb ischemia by ligation of the femoral artery in Townes SS (sickle cell) mice compared with AA (wild type) mice. Perfusion recovery, ascertained using LASER (light amplification by stimulated emission of radiation) Doppler perfusion imaging, showed significant diminution in collateral vessel formation in SS mice after hindlimb ischemia (76±13% AA versus 34±10% in SS by day 28; P<0.001; n=10 per group). The incidence of amputation (25% versus 5%) and foot necrosis (80% versus 15%) after hindlimb ischemia was significantly increased in the SS mice. Motor function recovery evaluation by the running wheel assay was also impaired in SS mice (36% versus 97% at 28 days post-hindlimb ischemia; P<0.001). This phenotype was associated with persistent and excessive production of reactive oxygen species by neutrophils. Importantly, neutrophil depletion or treatment with the antioxidant N-acetylcysteine reduced oxidative stress and improved functional collateral formation in the SS mice. CONCLUSIONS: Our data suggest dysfunctional collateral vessel formation in SS mice after vascular injury and provide a mechanistic basis for the multiple vascular complications of sickle cell disease.

Killing Me Unsoftly: Causes and Mechanisms of Arterial Stiffness.

The aorta is a blood vessel that provides a low-resistance path for blood flow directed from the heart to peripheral organs and tissues. However, the aorta has another central hemodynamic function, whereby the elastic nature of the aortic wall provides a significant biomechanical buffering capacity complementing the pulsatile cardiac blood flow, and this is often referred to as Windkessel function. Stiffening of the arterial wall leads to fundamental alterations in central hemodynamics, with widespread detrimental implications for organ function. In this Recent Highlights article, we describe recent contributions in ATVB that have highlighted the novel mechanisms and consequences of arterial stiffness and the clinical conditions in which arterial stiffness occurs, with a focus on advancements in the field.

Hydrogen peroxide regulates osteopontin expression through activation of transcriptional and translational pathways.

Recent in vivo studies establish that osteopontin (OPN) expression is hydrogen peroxide (H2O2)-dependent. However, the mechanisms by which H2O2 increases OPN expression remain poorly defined. OPN protein expression increased in an unusual biphasic pattern in response to H2O2. To investigate whether these increases were mediated through transcriptional and/or translational regulation of OPN, smooth muscle cells stimulated with 50 µM H2O2 were used as an in vitro cell system. Early protein increases at 6 h were not preceded by increased mRNA, whereas later increases (18 h) were, suggesting multiple mechanisms of regulation by H2O2. Polyribosomal fractionation assays established that early increases (6 h) in OPN expression were due to increased translation. This increase in translation occurred through phosphorylation of 4E-BP1 at the reactive oxygen species-sensitive Ser-65, which allowed for release and activation of eukaryotic initiation factor eIF4E and subsequent OPN translation. This early increase (6 h) in OPN was blunted in cells expressing a phospho-deficient 4E-BP1 mutant. H2O2 stimulation increased rat OPN promoter activity at 8 and 18 h, and promoter truncation studies established that promoter region -2284 to -795 is crucial for H2O2-dependent OPN transcription. ChIP studies determined that H2O2-dependent transcription is mediated by the reactive oxygen species-sensitive transcription factors NF-κB and AP-1. In conclusion, H2O2 stimulates OPN expression in a unique biphasic pattern, where early increases are translational and late increases are transcriptional.

Poldip2 controls vascular smooth muscle cell migration by regulating focal adhesion turnover and force polarization.

Polymerase-δ-interacting protein 2 (Poldip2) interacts with NADPH oxidase 4 (Nox4) and regulates migration; however, the precise underlying mechanisms are unclear. Here, we investigated the role of Poldip2 in focal adhesion turnover, as well as traction force generation and polarization. Poldip2 overexpression (AdPoldip2) in vascular smooth muscle cells (VSMCs) impairs PDGF-induced migration and induces a characteristic phenotype of long cytoplasmic extensions. AdPoldip2 also prevents the decrease in spreading and increased aspect ratio observed in response to PDGF and slightly impairs cell contraction. Moreover, AdPoldip2 blocks focal adhesion dissolution and sustains H2O2 levels in focal adhesions, whereas Poldip2 knockdown (siPoldip2) significantly decreases the number of focal adhesions. RhoA activity is unchanged when focal adhesion dissolution is stimulated in control cells but increases in AdPoldip2-treated cells. Inhibition of RhoA blocks Poldip2-mediated attenuation of focal adhesion dissolution, and overexpression of RhoA or focal adhesion kinase (FAK) reverses the loss of focal adhesions induced by siPoldip2, indicating that RhoA and FAK mediate the effect of Poldip2 on focal adhesions. Nox4 silencing prevents focal adhesion stabilization by AdPoldip2 and induces a phenotype similar to siPoldip2, suggesting a role for Nox4 in Poldip2-induced focal adhesion stability. As a consequence of impaired focal adhesion turnover, PDGF-treated AdPoldip2 cells are unable to reduce and polarize traction forces, a necessary first step in migration. These results implicate Poldip2 in VSMC migration via regulation of focal adhesion turnover and traction force generation in a Nox4/RhoA/FAK-dependent manner.

Reactive oxygen species regulate osteopontin expression in a murine model of postischemic neovascularization.

OBJECTIVE: Previous findings from our laboratory demonstrated that neovascularization was impaired in osteopontin (OPN) knockout animals. However, the mechanisms responsible for the regulation of OPN expression in the setting of ischemia remain undefined. Therefore, we sought to determine whether OPN is upregulated in response to ischemia and hypothesized that hydrogen peroxide (H(2)O(2)) is a critical component of the signaling mechanism by which OPN expression is upregulated in response to ischemia in vivo. METHODS AND RESULTS: To determine whether ischemic injury upregulates OPN, we used a murine model of hindlimb ischemia. Femoral artery ligation in C57BL/6 mice significantly increased OPN expression and H(2)O(2) production. Infusion of C57BL/6 mice with polyethylene glycol-catalase (10 000 U/kg per day) or the use of transgenic mice with smooth muscle cell-specific catalase overexpression blunted ischemia-induced OPN, suggesting ischemia-induced OPN expression is H(2)O(2)-dependent. Decreased H(2)O(2)-mediated OPN blunted reperfusion and collateral formation in vivo. In contrast, the overexpression of OPN using lentivirus restored neovascularization. CONCLUSIONS: Scavenging H(2)O(2) blocks ischemia-induced OPN expression, providing evidence that ischemia-induced OPN expression is H(2)O(2) dependent. Decreased OPN expression impaired neovascularization, whereas overexpression of OPN increased angiogenesis, supporting our hypothesis that OPN is a critical mediator of postischemic neovascularization and a potential novel therapeutic target for inducing new vessel growth.

Current State of Pediatric Reference Intervals and the Importance of Correctly Describing the Biochemistry of Child Development: A Review.

Importance: Appropriately established pediatric reference intervals are critical to the clinical decision-making process and should reflect the physiologic changes that occur during healthy child development. Reference intervals used in pediatric care today remain highly inconsistent across a broad range of common clinical biomarkers. Observations: This narrative review assesses biomarker-specific pediatric reference intervals and their clinical utility with respect to the underlying biological changes occurring during development. Pediatric reference intervals from PubMed-indexed articles published from January 2015 to April 2021, commercial laboratory websites, study cohorts, and pediatric reference interval books were all examined. Although large numbers of pediatric reference intervals are published for some biomarkers, very few are used by clinical and commercial laboratories. The patterns, extent, and timing of biomarker changes are highly variable, particularly during developmental stages with rapid physiologic changes. However, many pediatric reference intervals do not capture these changes and thus do not accurately reflect the underlying biochemistry of development, resulting in significant inconsistencies between reference intervals. Conclusions and Relevance: There is a need to correctly describe the biochemistry of child development as well as to identify strategies to develop accurate and consistent pediatric reference intervals for improved pediatric care.

Poldip2, a novel regulator of Nox4 and cytoskeletal integrity in vascular smooth muscle cells.

RATIONALE: NADPH oxidases (Noxes) regulate vascular physiology and contribute to the pathogenesis of vascular disease. In vascular smooth muscle cells (VSMCs), the interactions of individual Nox homologs with regulatory proteins are poorly defined. OBJECTIVE: The objective of this study was to identify novel NADPH oxidase regulatory proteins. METHODS AND RESULTS: Using a yeast 2-hybrid screen, we identified a novel p22phox binding partner, Poldip2, and demonstrated that it associates with p22phox, NADPH oxidase (Nox)1, and Nox4 and colocalizes with p22phox at sites of Nox4 localization. Poldip2 increases Nox4 enzymatic activity by 3-fold and positively regulates basal reactive oxygen species production in VSMCs (O2(.-): 86.3+/-15.6% increase; H2O2: 40.7+/-4.5% increase). Overexpression of Poldip2 activates Rho (180.2+/-24.8% increase), strengthens focal adhesions, and increases stress fiber formation. These phenotypic changes are blocked by dominant negative Rho. In contrast, depletion of either Poldip2 or Nox4 results in a loss of these structures, which is rescued by adding back active Rho. Cell migration, which requires dynamic cytoskeletal remodeling, is impaired by either excess (70.1+/-14.7% decrease) or insufficient Poldip2 (63.5+/-5.9% decrease). CONCLUSIONS: These results suggest that Poldip2 associates with p22phox to activate Nox4, leading to regulation of focal adhesion turnover and VSMC migration, thus linking reactive oxygen species production and cytoskeletal remodeling. Poldip2 may be a novel therapeutic target for vascular pathologies with a significant VSMC migratory component, such as restenosis and atherosclerosis.

NADPH Oxidases and Measurement of Reactive Oxygen Species.

The NADPH oxidase (Nox) family of enzymes is expressed in many tissues that are involved in hypertension, including blood vessels, kidney, and brain. In these tissues, the products of NADPH oxidase activity, superoxide and ultimately hydrogen peroxide, act as intracellular and extracellular messengers during compartmentalized cellular signaling. The correct measurement of Nox activity and its products is crucial to enable studies of how these signaling pathways affect the molecular mechanisms underlying hypertension. Here, we describe methods for detection and measurement of hydrogen peroxide and superoxide derived from NADPH oxidases in biological samples such as cells and tissues.

A Novel Technique for Accelerated Culture of Murine Mesenchymal Stem Cells that Allows for Sustained Multipotency.

Bone marrow derived mesenchymal stem cells (MSCs) are regularly utilized for translational therapeutic strategies including cell therapy, tissue engineering, and regenerative medicine and are frequently used in preclinical mouse models for both mechanistic studies and screening of new cell based therapies. Current methods to culture murine MSCs (mMSCs) select for rapidly dividing colonies and require long-term expansion. These methods thus require months of culture to generate sufficient cell numbers for feasibility studies in a lab setting and the cell populations often have reduced proliferation and differentiation potential, or have become immortalized cells. Here we describe a simple and reproducible method to generate mMSCs by utilizing hypoxia and basic fibroblast growth factor supplementation. Cells produced using these conditions were generated 2.8 times faster than under traditional methods and the mMSCs showed decreased senescence and maintained their multipotency and differentiation potential until passage 11 and beyond. Our method for mMSC isolation and expansion will significantly improve the utility of this critical cell source in pre-clinical studies for the investigation of MSC mechanisms, therapies, and cell manufacturing strategies.