Synergism of red blood cells and tranexamic acid in the inhibition of fibrinolysis.

BACKGROUND: Postpartum hemorrhage (PPH) is the leading cause of maternal death worldwide. The World Maternal Antifibrinolytic trial showed that antifibrinolytic tranexamic acid (TXA) reduces PPH deaths. Maternal anemia increases the risk of PPH. The World Maternal Antifibrinolytic-2 trial is now assessing whether TXA can prevent PPH in women with anemia. Low red blood cell (RBC) counts promote fibrinolysis by altering fibrin structure and plasminogen activation. OBJECTIVES: We explored interactions between RBCs and TXA in inhibiting fibrinolysis. METHODS: We used global fibrinolytic assays (ball sedimentation and viscoelasticity) to monitor the lysis of fibrin containing plasminogen and tissue-type plasminogen activator. We applied a fluorogenic kinetic assay to measure plasmin generation in fibrin clots and scanning electron microscopy to study fibrin structure. RESULTS: According to parallel-line bioassay analysis of the fibrin lysis-time data, the antifibrinolytic potency of 4-128 µM TXA was increased in the presence of 10% to 40% (v/v) RBCs. Global fibrinolysis assays showed that the joint effect of RBCs and TXA was about 15% larger than the sum of their individual effects in the inhibition of fibrinolysis. In plasminogen activation, TXA added the same increment of inhibition to the effect of RBCs at any cell count in the fibrin clot. Regarding fibrin structure, TXA thickened fibrin fibers, which impaired plasminogen activation, whereas RBCs promoted fine fibers that were more resistant to plasmin. CONCLUSIONS: The antifibrinolytic potency of TXA is enhanced in fibrin formed in the presence of RBCs through inhibition of plasminogen activation and fibrin lysis, which correlates with modifications of fibrin structures.

Sources of bias and limitations of thrombinography: inner filter effect and substrate depletion at the edge of failure algorithm.

BACKGROUND: Fluorogenic thrombin generation (TG) is a global hemostasis assay that provides an overall representation of hemostasis potential. However, the accurate detection of thrombin activity in plasma may be affected by artifacts inherent to the assay-associated fluorogenic substrate. The significance of the fluorogenic artifacts or their corrections has not been studied in hemophilia treatment applications. METHODS: We sought to investigate TG in hemophilia plasma samples under typical and worst-case fluorogenic artifact conditions and assess the performance of artifact correction algorithms. Severe hemophilic plasma with or without added Factor VIII (FVIII) was evaluated using commercially available and in-house TG reagents, instruments, and software packages. The inner filter effect (IFE) was induced by spiking elevated amounts of fluorophore 7-amino-4-methylcoumarin (AMC) into plasma prior to the TG experiment. Substrate consumption was modeled by adding decreasing amounts of Z-Gly-Gly-Arg-AMC (ZGGR-AMC) to plasma or performing TG in antithrombin deficient plasma. RESULTS: All algorithms corrected the AMC-induced IFE and antithrombin-deficiency induced substrate consumption up to a certain level of either artifact (edge of failure) upon which TG results were not returned or overestimated. TG values in FVIII deficient (FVIII-DP) or supplemented plasma were affected similarly. Normalization of FVIII-DP resulted in a more accurate correction of substrate artifacts than algorithmic methods. CONCLUSIONS: Correction algorithms may be effective in situations of moderate fluorogenic substrate artifacts inherent to highly procoagulant samples, but correction may not be required under typical conditions for hemophilia treatment studies if TG parameters can be normalized to a reference plasma sample.

Glycated albumin modulates the contact system with implications for the kallikrein-kinin and intrinsic coagulation systems.

BACKGROUND: Human serum albumin (HSA) is the most abundant plasma protein and is sensitive to glycation in vivo. The chronic hyperglycemic conditions in patients with diabetes mellitus (DM) induce a nonenzymatic Maillard reaction that denatures plasma proteins and forms advanced glycation end products (AGEs). HSA-AGE is a prevalent misfolded protein in patients with DM and is associated with factor XII activation and downstream proinflammatory kallikrein-kinin system activity without any associated procoagulant activity of the intrinsic pathway. OBJECTIVES: This study aimed to determine the relevance of HSA-AGE toward diabetic pathophysiology. METHODS: The plasma obtained from patients with DM and euglycemic volunteers was probed for activation of FXII, prekallikrein (PK), and cleaved high-molecular-weight kininogen by immunoblotting. Constitutive plasma kallikrein activity was determined via chromogenic assay. Activation and kinetic modulation of FXII, PK, FXI, FIX, and FX via in vitro-generated HSA-AGE were explored using chromogenic assays, plasma-clotting assays, and an in vitro flow model using whole blood. RESULTS: Plasma obtained from patients with DM contained increased plasma AGEs, activated FXIIa, and resultant cleaved cleaved high-molecular-weight kininogen. Elevated constitutive plasma kallikrein enzymatic activity was identified, which positively correlated with glycated hemoglobin levels, representing the first evidence of this phenomenon. HSA-AGE, generated in vitro, triggered FXIIa-dependent PK activation but limited the intrinsic coagulation pathway activation by inhibiting FXIa and FIXa-dependent FX activation in plasma. CONCLUSION: These data indicate a proinflammatory role of HSA-AGEs in the pathophysiology of DM via FXII and kallikrein-kinin system activation. A procoagulant effect of FXII activation was lost through the inhibition of FXIa and FIXa-dependent FX activation by HSA-AGEs.

Citrullinated fibrinogen forms densely packed clots with decreased permeability.

BACKGROUND: Fibrin, the main scaffold of thrombi, is susceptible to citrullination by PAD (peptidyl arginine deiminase) 4, secreted from neutrophils during the formation of neutrophil extracellular traps. Citrullinated fibrinogen (citFg) has been detected in human plasma as well as in murine venous thrombi, and it decreases the lysability and mechanical resistance of fibrin clots. OBJECTIVE: To investigate the effect of fibrinogen citrullination on the structure of fibrin clots. METHODS: Fibrinogen was citrullinated with PAD4 and clotted with thrombin. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) were used to measure fiber thickness, fiber height/width ratio, and fiber persistence length in clots containing citFg. Fiber density was measured with laser scanning microscopy (LSM) and permeability measurements were carried out to estimate the porosity of the clots. The intra-fiber structure of fibrin was analyzed with small-angle X-ray scattering (SAXS). RESULTS: SEM images revealed a decrease in the median fiber diameter that correlated with the fraction of citFg in the clot, while the fiber width/length ratio remained unchanged according to AFM. With SAXS we observed that citrullination resulted in the formation of denser clots in line with increased fiber density shown by LSM. The permeability constant of citrullinated fibrin decreased more than 3-fold indicating significantly decreased porosity. SAXS also showed largely preserved periodicity in the longitudinal assembly of fibrin monomers. CONCLUSION: The current observations of thin fibers combined with dense packing and low porosity in the presence of citFg can provide a structural framework for the mechanical fragility and lytic resistance of citrullinated fibrin.

Is it possible to make a common reference standard for D-dimer measurements? Communication from the ISTH SSC Subcommittee on Fibrinolysis.

BACKGROUND: D-dimer antigen is a heterogeneous mixture of fibrin degradation products that when present at high levels in plasma indicate ongoing coagulation and fibrinolysis. The heterogeneous nature of the target D-dimer antigen and the variety of assay systems means that it is difficult to compare results from different methods. OBJECTIVES: To identify a universally agreed D-dimer standard that could help harmonize results from different methods. METHODS: A pool of patient plasma with high D-dimer levels was freeze-dried and investigated as a long-term World Health Organization international standard for D-dimer. Fibrin degradation products from clot lysis reactions were also freeze-dried in various formulations and investigated in commutability studies with patient plasma. RESULTS: Problems of instability of D-dimer plasma emerged suggesting loss of reactivity after freeze-drying and storage at -20°C of 10%-18% per year. Freeze-dried fibrin degradation products added to plasma were also unstable, but the sugar trehalose was found to improve stability. However, this preparation was not suitable as a standard in widely used assay platforms. Previous studies suggest fibrin degradation products are prone to structural rearrangements and amyloid formation, which may explain the instability of candidate D-dimer standards. CONCLUSIONS: The known difficulties of D-dimer standardization are compounded by instability of D-dimer antigen after freeze-drying, described in this report. Fibrin degradation products added to plasma and stabilized by trehalose are not suitable as a standard for D-dimer measurement harmonization. Trehalose stabilization of pooled patient plasma containing high D-dimer levels may produce a useful standard, but this requires confirmation.

Fibrinogen-mimicking, multiarm nanovesicles for human thrombus-specific delivery of tissue plasminogen activator and targeted thrombolytic therapy.

Clinical use of tissue plasminogen activator (tPA) in thrombolytic therapy is limited by its short circulation time and hemorrhagic side effects. Inspired by fibrinogen binding to activated platelets, we report a fibrinogen-mimicking, multiarm nanovesicle for thrombus-specific tPA delivery and targeted thrombolysis. This biomimetic system is based on the lipid nanovesicle coated with polyethylene glycol (PEG) terminally conjugated with a cyclic RGD (cRGD) peptide. Our experiments with human blood demonstrated its highly selective binding to activated platelets and efficient tPA release at a thrombus site under both static and physiological flow conditions. Its clot dissolution time in a microfluidic system was comparable to that of free tPA. Furthermore, we report a purpose-built computational model capable of simulating targeted thrombolysis of the tPA-loaded nanovesicle and with a potential in predicting the dynamics of thrombolysis in physiologically realistic scenarios. This combined experimental and computational work presents a promising platform for development of thrombolytic nanomedicines.

An international collaborative study to establish the WHO 3rd International Standard for Thrombin: Communication from the ISTH SSC subcommittee on factor XIII and fibrinogen.

The calibration of thrombin products relies on the World Health Organization (WHO) 2nd International Standard (IS) for Thrombin (01/580) which defines the international unit (IU) for thrombin potency. With stocks of the 2nd IS (01/580) running low, an international collaborative study was organized to calibrate a replacement. Twenty laboratories from 13 countries took part in the study and measured the potency of two candidate replacement standards (coded 01/578 and 19/188) relative to the 2nd IS. In total, 111 valid assays were returned, which were a combination of plasma/fibrinogen clotting assays and chromogenic assays. Variation between and within laboratories was low, with inter- and intra-laboratory geometric coefficient of variation (GCV) generally <5% for all assay methods and substrates. For 01/578, potency estimates by clotting assays (101.1 IU/ampoule) were significantly lower than estimates by chromogenic assays (111.5 IU/ampoule). Mean potency estimates for 19/188 were 90.4 IU/ampoule by clotting assay and 88.1 IU/ampoule by chromogenic assay, which was not a statistically significant difference. The close ratio between clotting and chromogenic assay potency estimates for 19/188 suggests it has a higher α-thrombin content than 01/578 and is equivalent to the current IS (01/580). Accelerated degradation studies predicted excellent long-term stability profiles for preparations 01/580, 01/578, and 19/188. Based on the results of this study, the WHO Expert Committee on Biological Standardization established 19/188 as the 3rd IS for Thrombin with a potency of 90 IU/ampoule in August 2020.

Measuring Fibrinolysis.

Physiological fibrinolysis under normal conditions progresses slowly, in contrast to coagulation which is triggered rapidly to stop bleeding and defend against microbial invasion. Methods to detect fibrinolysis abnormalities are less simple and poorly standardized compared with common coagulation tests. Fibrinolysis can be accelerated by preparing euglobulin from plasma to reduce endogenous inhibitors, or by adding plasminogen activators to normal plasma. However, these manipulations complicate interpretation of results and diagnosis of a "fibrinolysis deficit." Many observational studies on antigen levels of fibrinolysis inhibitors, plasminogen activator inhibitor 1 or thrombin-activatable fibrinolysis inhibitor, zymogen or active enzyme have been published. However, conclusions are mixed and there are clear problems with harmonization of results. Viscoelastic methods have the advantage of being rapid and are used as point-of-care tests. They also work with whole blood, allowing the contribution of platelets to be explored. However, there are no agreed protocols for applying viscoelastic methods in acute care for the diagnosis of hyperfibrinolysis or to direct therapy. The emergence of SARS-CoV-2 and the dangers of associated coagulopathy provide new challenges. A common finding in hospitalized patients is high levels of D-dimer fibrin breakdown products, indicative of ongoing fibrinolysis. Well-established problems with D-dimer testing standardization signal that we should be cautious in using results from such tests as prognostic indicators or to target therapies.

Size- and charge-dependent modulation of the lytic susceptibility and mechanical stability of fibrin-histone clots by heparin and polyphosphate variants.

BACKGROUND: Neutrophil extracellular traps (NETs) containing DNA and histones are expelled from neutrophils in infection and thrombosis. Heparins, anticoagulant polyanions, can neutralize histones with a potential therapeutic advantage in sepsis. Polyphosphates, procoagulant polyanions, are released by platelets and microorganisms. OBJECTIVES: To characterize the combined effects of NET components and polyanions on clot structure, mechanical properties and lytic susceptibility. METHODS: Scanning electron microscopy, pressure-driven permeation, turbidimetry, and oscillation rheometry were used for the characterization of the structure, viscoelasticity, and kinetics of formation and lysis of fibrin and plasma clots containing histones+/-DNA in combination with unfractionated heparin, its desulfated derivatives, low molecular weight heparin (LMWH), pentasaccharide, and polyphosphates of different sizes. RESULTS: Histones and DNA inhibited fibrin lysis by plasmin, but this behavior was not neutralized by negatively charged heparins or short polyphosphates. Rather, fibrin lysis was further inhibited by added polyanions. Histones inhibited plasma clot lysis by tissue plasminogen activator and the response to added heparin was size dependent. Unfractionated heparin, LMWH, and pentasaccharide had no effect, exacerbated, or reversed histone inhibition, respectively. Histones increased the mechanical strength of fibrin, which was exacerbated by smaller heparin and polyphosphate molecules. Histones increased fibrin diameter and pore size of fibrin clots and this effect was neutralized by all heparin variants but enhanced by polyphosphates. CONCLUSIONS: Despite their common polyanionic character, heparins and polyphosphates exert distinct effects on fibrin mechanical and fibrinolytic stability. Anti-fibrinolytic effects of histones were more often enhanced by polyanions not counteracted. Careful selection of anti-histone strategies is required if they are to be combined with thrombolytic therapy.

Kallikrein directly interacts with and activates Factor IX, resulting in thrombin generation and fibrin formation independent of Factor XI.

Kallikrein (PKa), generated by activation of its precursor prekallikrein (PK), plays a role in the contact activation phase of coagulation and functions in the kallikrein-kinin system to generate bradykinin. The general dogma has been that the contribution of PKa to the coagulation cascade is dependent on its action on FXII. Recently this dogma has been challenged by studies in human plasma showing thrombin generation due to PKa activity on FIX and also by murine studies showing formation of FIXa-antithrombin complexes in FXI deficient mice. In this study, we demonstrate high-affinity binding interactions between PK(a) and FIX(a) using surface plasmon resonance and show that these interactions are likely to occur under physiological conditions. Furthermore, we directly demonstrate dose- and time-dependent cleavage of FIX by PKa in a purified system by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and chromogenic assays. By using normal pooled plasma and a range of coagulation factor-deficient plasmas, we show that this action of PKa on FIX not only results in thrombin generation, but also promotes fibrin formation in the absence of FXII or FXI. Comparison of the kinetics of either FXIa- or PKa-induced activation of FIX suggest that PKa could be a significant physiological activator of FIX. Our data indicate that the coagulation cascade needs to be redefined to indicate that PKa can directly activate FIX. The circumstances that drive PKa substrate specificity remain to be determined.

Development of an updated assay for prekallikrein activator in albumin and immunoglobulin therapeutics.

BACKGROUND: Prekallikrein activator (PKA) is a contaminating enzyme found in therapeutic albumin and immunoglobulin products. The level is commonly measured using methods such as that defined by the European Pharmacopoeia (Ph Eur) with traceability to the WHO International Standard for PKA. This method generally works well, but problems are sometimes observed. MATERIALS AND METHODS: A simplified one-step method has been developed to replace the existing Ph Eur two-step method which consists of kallikrein generation followed by kallikrein measurement using a chromogenic substrate. Analysis of data from the one-stage method is simplified by the use of a dedicated online app. RESULTS: The one-stage method was validated against the current Ph Eur method using batches of albumin and immunoglobulins. Problem batches of immunoglobulins were investigated using the one-stage method. Improved methodology using true initial rate determinations and use of acid-treated prekallikrein substrate (PKS) helped understand and reduce artefactual results. CONCLUSIONS: The one-stage method and associated app streamline real-time determination of PKA and promote good principles of enzyme assays to limit substrate depletion, while also conserving expensive PKS. Blanking steps and reproducibility are simplified.

Extracellular Histones Inhibit Fibrinolysis through Noncovalent and Covalent Interactions with Fibrin.

Histones released into circulation as neutrophil extracellular traps are causally implicated in the pathogenesis of arterial, venous, and microvascular thrombosis by promoting coagulation and enhancing clot stability. Histones induce structural changes in fibrin rendering it stronger and resistant to fibrinolysis. The current study extends these observations by defining the antifibrinolytic mechanisms of histones in purified, plasma, and whole blood systems. Although histones stimulated plasminogen activation in solution, they inhibited plasmin as competitive substrates. Protection of fibrin from plasmin digestion is enhanced by covalent incorporation of histones into fibrin, catalyzed by activated transglutaminase, coagulation factor FXIII (FXIIIa). All histone subtypes (H1, H2A, H2B, H3, and H4) were crosslinked to fibrin. A distinct, noncovalent mechanism explains histone-accelerated lateral aggregation of fibrin protofibrils, resulting in thicker fibers with higher mass-to-length ratios and in turn hampered fibrinolysis. However, histones were less effective at delaying fibrinolysis in the absence of FXIIIa activity. Therapeutic doses of low-molecular-weight heparin (LMWH) prevented covalent but not noncovalent histone-fibrin interactions and neutralized the effects of histones on fibrinolysis. This suggests an additional antithrombotic mechanism for LMWH beyond anticoagulation. In conclusion, for the first time we report that histones are crosslinked to fibrin by FXIIIa and promote fibrinolytic resistance which can be overcome by FXIIIa inhibitors and histone-binding heparinoids. These findings provide a rationale for targeting the FXIII-histone-fibrin axis to destabilize fibrin and prevent potentially thrombotic fibrin networks.

Fibrinogen protects neutrophils from the cytotoxic effects of histones and delays neutrophil extracellular trap formation induced by ionomycin.

Neutrophils are pivotal players in immune defence which includes a process of release of histones and DNA as neutrophil extracellular traps (NETs). Histones, while toxic to invading pathogens, also kill host cells, including neutrophils. Bacteria have evolved mechanisms to escape neutrophils, including the secretion of leucocidins (e.g. ionomycin). Live cell video microscopy showed how fibrinogen and fibrin influence NETosis and neutrophil responses to extracellular histones. Histones were rapidly lethal to neutrophils after binding to cells, but formation of fibrinogen/fibrin-histone aggregates prevented cell death. Histone cytotoxicity was also reduced by citrullination by peptidyl arginine deiminase 4, or digestion by serine proteases. Ionomycin and phorbol 12-myristate 13 acetate (PMA) are used to trigger NETosis. Fibrinogen was responsible for a second distinct mechanism of neutrophil protection after treatment with ionomycin. Fibrinogen clustered on the surface of ionomycin-stimulated neutrophils to delay NETosis; and blocking the ß integrin receptor, αMß2, abolished fibrinogen protection. Fibrinogen did not bind to or protect neutrophils stimulated with PMA. Fibrinogen is an acute phase protein that will protect exposed cells from damaging circulating histones or leucocidins; but fibrinogen depletion/consumption, as in trauma or sepsis will reduce protection. It is necessary to consider the role of fibrinogen in NETosis.

An international collaborative study to establish the WHO 4th International Standard for Streptokinase: Communication from the SSC of the ISTH.

Streptokinase is used worldwide as a cost-effective treatment for acute myocardial infarction. Manufacturers use the World Health Organization (WHO) International Standard (IS) for Streptokinase to potency label their products, ensuring consistent, safe, and effective dosing. Stocks of the third IS for streptokinase (coded 00/464) are running low, and an international collaborative study was organized to calibrate a replacement. A total of 15 laboratories from nine countries took part, using chromogenic and/or fibrin clot lysis methods to determine the potency of two candidate preparations, coded 16/356 (sample B) and 16/358 (sample C), relative to the third IS (00/464). A third sample (88/824, sample A), which was used in the collaborative studies to establish the second and third IS, was also included. There was good agreement in potency estimates from different assay methods and low variability both within and between laboratories. Long-term stability modeling indicated the candidates are very stable. Comparison of potency estimates for 88/824 (sample A) with potencies calculated in previous studies revealed a variability of only 1.9% over the course of three collaborative studies spanning 30 years and more than 50 years of streptokinase standardization. This indicates excellent continuity of the International Unit (IU) and assay methods. Following agreement by study participants and Scientific and Standardization Committee experts of the International Society on Thrombosis and Haemostasis, the WHO Expert Committee on Biological Standardization established 16/358 (sample C) as the fourth IS for Streptokinase with a potency of 1013 IU per ampoule in October 2019.

Biorelevant polyanions stabilize fibrin against mechanical and proteolytic decomposition: Effects of polymer size and electric charge.

The release of neutrophil extracellular traps (NETs) containing DNA and histones is an essential mechanism in the neutrophil-mediated innate immunity. In thrombi the polyanionic DNA confers mechanical and lytic resistance to fibrin and heparins interfere with the effects of NET components. Heparins are polyanions used not only as therapeutic agents, but they are also released by mast cells at entry sites of pathogens. Platelets and microorganisms release a different type of polyanions (polyphosphates) of various size (in the range 60-1000 phosphate monomers). With the current study we aimed to evaluate if the stability of fibrin is influenced by the type of polyanion, its molecular size or relative electric charge. Fibrin structure was approached with scanning electron microscopy (SEM) and pressure-driven permeation. An oscillation rheometer was used to investigate viscoelastic properties. Kinetic turbidimetric assays for the generation and dissolution of composite fibrin clots containing unfractionated heparin (UFH), and its partially or fully desulfated derivatives, as well as low molecular-weight heparin (LMWH), pentasaccharide (S5), and polyphosphates composed of 45 (P45), 100 (P100) or 700 (P700) monomers at average. The smaller polyanions P45, P100, LMWH, and S5 accelerated, whereas P700 and UFH retarded clot formation. All polyanions altered the fibrin structure: SEM and clot permeation showed thicker fibers with smaller (LMWH, S5, P700) or larger (UFH, P100) pores. All polyanions stabilized the clots mechanically, but the smaller P45, P100 and LMWH decreased the deformability of fibrin, whereas the large UFH and P700 increased the maximal bearable deformation of clots. Despite the size-dependent structural changes, all heparins caused a 10-15% prolongation of lysis-times with plasmin, and UFH-effects depended on sulfation patterns. The 20-35% prolongation of lysis-times caused by all polyphosphates was a kringle-dependent phenomenon, and was dampened in the presence of 6-aminohexanoate blocking the lysine-binding sites of plasmin. In summary, we found that polyanions of different chemical structure stabilize fibrin clots via size-dependent modulation of fibrin structure and kringle-dependent inhibition of plasmin-mediated fibrinolysis.

Computational simulations of thrombolysis in acute stroke: Effect of clot size and location on recanalisation.

Acute ischaemic stroke can be treated by intravenous thrombolysis whereby tissue plasminogen activator (tPA) is infused to dissolve clots that block blood supply to the brain. In this study, we aim to examine the influence of clot location and size on lysis pattern and recanalisation by using a recently developed computational modelling framework for thrombolysis under physiological flow conditions. An image-based patient-specific model is reconstructed which consists of the internal carotid bifurcation with the A1 segment of anterior cerebral arteries and M1 segment of middle cerebral arteries, and the M1 bifurcation containing the M2 segments. By varying the clot size and location, 7 scenarios are simulated mimicking thrombolysis of M1 and M2 occlusions. Our results show that initial breakthrough always occurs along the inner curvature of the occluded cerebral artery, due to prolonged tPA residence time in the recirculation zone. For a given occlusion site, lysis completion time appears to increase almost quadratically with the initial clot volume; whereas for a given clot volume, the simulated M2 occlusions take up to 30% longer for complete lysis compared to the corresponding M1 occlusions.

Applying the science of measurement to biology: Why bother?

Both basic and translational research are continuously evolving, but the principles that underpin research integrity remain constant. These include rational, hypothesis-driven, and adequately planned and controlled science, which is carried out openly, honestly, and ethically. An important component of this should be minimising experimental irreproducibility. Biological systems, in particular, are inherently variable due to the nature of cells and tissues, as well as the complex molecules within them. As a result, it is important to understand and identify sources of variability and to strive to minimise their influence. In many instances, the application of metrology (the science of measurement) can play an important role in ensuring good quality research, even within biological systems that aren't always amenable to many of the metrological concepts applied in other fields. Here, we introduce the basic concepts of metrology in relation to biological systems and promote the application of these principles to help avoid potentially costly mistakes in both basic and translational research. We also call on funders to encourage the uptake of metrological principles, as well as provide funding and support for later engagement with regulatory bodies.