In Support of Venous Glucose as a Reference Matrix for Evaluating Continuous Glucose Monitoring Accuracy

Introduction

Continuous glucose monitoring (CGM) is standard of care in people with diabetes who are taking insulin, and it is growing in use among people not on insulin therapy. It is the duty of our regulatory bodies to ensure the accuracy of commercially available CGM systems. Knowing that these devices provide accurate glucose results is critical for patients and providers.

A fundamental part of testing protocols for assessing the accuracy of CGM systems has recently been challenged. CGM performance studies currently compare glucose readings from these sensors with glucose readings from blood specimens obtained from venous or capillary sources, typically obtained, respectively, through multiple draws from an indwelling intravenous line ¹ or multiple fingerstick specimens. ² The regulatory bodies determining approval in the United States (Food and Drug Administration [FDA]) ³ and Europe (The Conformité Européenne [CE]) ⁴ permit blood from either source to be used as a comparator (provided the comparator method is a well-validated laboratory procedure, and the study design is appropriate and consistent with regulatory standards). Reference testing based on venous blood is widely used in the United States for testing the accuracy of CGM systems.

The FDA permits venous blood testing provided it is measured on an FDA-accepted laboratory comparator under the integrated continuous glucose monitoring system (iCGM) special controls, which are device-specific requirements (beyond general controls) that an iCGM must meet for class II classification. Class II devices are considered moderate-to-high risk and are controlled by both general controls and special controls, while Class III devices are high risk and generally require more extensive evidence of safety and effectiveness. The FDA defines an iCGM as a CGM that reliably and securely transmits glucose data to digitally connected devices, including automated insulin dosing systems. ⁵ In a pair of 2025 reports by the International Federation of Clinical Chemistry and Laboratory Medicine Working Group (IFCC WG) on CGM, the majority of group members recommended that capillary samples (rather than venous samples) be considered the preferred reference matrix.^6,7 This Working Group (WG) has made thoughtful and comprehensive recommendations for testing CGM systems for accuracy, and aims to align their recommendations with future International Organization for Standardization (ISO) standards. ⁸

Comparator blood matrix selection is a critical design element of accuracy studies. The matrix selected affects the testing process, the observed performance, and our ability to compare results across studies. In this commentary, we present the position that (1) venous blood (along with capillary blood) should be considered as a legitimate reference matrix for comparator glucose testing for regulatory submissions and clinical evaluations, and (2) venous blood should not be considered less preferred than capillary blood. This position is in contrast to the IFCC WG’s recommendation for use of capillary samples where the majority of WG-CGM members expressed a preference for the capillary sample origin, citing its practical relevance and historical use in self-monitoring practices of people with diabetes.

How CGMs are Tested for Accuracy With Capillary and Venous Blood as Comparator Samples

Currently in the United States, CGMs are generally tested for accuracy of glucose measurement using venous blood as a comparator matrix, whereas capillary blood as a comparator matrix is used for subjects with safety-related limitations on blood draw volume, such as pediatrics under age 6. ⁹ Several laboratory-based analyzers have been cleared and can be used as comparators to support regulatory submissions of glucose monitors seeking clearance for commercial use. Three examples are the YSI 2300 STAT Plus cleared by the FDA on October 2, 1991, ¹⁰ the Nova Primary Glucose Analyzer cleared by the FDA on October 22, 2022, ¹¹ and the YSI 2900C Biochemistry Analyzer cleared by FDA on September 24, 2024. ¹² All three use a 25-microliter sample of whole blood or plasma. All three are cleared to test venous whole blood or plasma samples, and the YSI 2900C Biochemistry Analyzer is also cleared to test capillary blood (ie, a fingerstick capillary blood sample drawn into K3EDTA microtubes, then analyzed). For the YSI 2300 STAT Plus and the Nova Primary Glucose Analyzer, using capillary blood, from a labeling and clearance standpoint, is protocol-specific and off-label rather than an on-label cleared capillary blood or capillary-plasma indication. Other laboratory analyzers may be acceptable if they are well-validated, demonstrate appropriate precision and bias, and are traceable to higher-order reference standards (eg, National Institute of Standards and Technology [NIST]) in accordance with FDA and international guidance on comparator methods. ¹³

In formal accuracy studies and regulatory submissions of CGMs, venous plasma measured on a laboratory analyzer that is cleared for this purpose has commonly been used as the primary reference. Glucose can be measured simultaneously in capillary and venous samples across a range of values and fit into regression models or bias can be computed to derive conversion equations between these two matrices. Validated equations can be prespecified in protocols and applied in analyses, so that capillary measurements can be transformed to venous plasma–equivalent values for regulatory evaluation.

An iCGM is an FDA Class II CGM that meets strict accuracy standards and interoperates with connected devices like automated insulin delivery systems. For all currently FDA-cleared iCGM systems, the pivotal trials have used venous blood (or arterialized-venous blood which is venous blood from a limb warmed to increase local blood flow and oxygenation) as the primary comparator matrix for subjects over six years of age. Advantages and disadvantages of capillary versus venous blood as comparator methods for CGM accuracy testing are presented in Table 1.

Table 1.

Advantages and Disadvantages of Capillary Versus Venous Blood as Comparator Methods for Testing CGM Accuracy.

Aspect	Capillary blood	Venous blood
Physiological alignment with CGM	Closer alignment with interstitial glucose, especially during rapid glucose excursions (postprandial changes, hypoglycemia).	Less physiologically aligned with interstitial fluid during rapid changes
Historical use	Historically used for CGM calibration, which can improve apparent agreement with glucose meters.	Widely used in US CGM accuracy studies as the comparator matrix.
Apparent CGM accuracy (MARD)	May yield a lower MARD, largely because of matching the calibration matrix rather than true superiority.	Venous versus capillary blood may show a higher MARD, but values from venous and capillary blood can be transformed from one to another
Analytical method quality	Often measured using point-of-care glucose meters, which are generally less accurate than laboratory-grade analyzers.	Measured using laboratory-grade analyzers (eg, YSI/Nova) with higher traceability, precision and accuracy.
Metrological traceability	Limited; whole blood harmonization is challenging to higher-order standards because whole blood NIST primary standards do not exist.	Strong; results can be recalibrated to NIST-certified targets and harmonized across methods.
Preanalytical variability	Higher variability due to tissue fluid contamination, inadequate wiping, and variable squeezing.	Lower variability: glycolysis can be minimized via an ice bath, glycolysis inhibitor tubes, such as citrate buffer tubes or sodium fluoride/potassium oxalate gray top tubes. and/or rapid separation of plasma from cells
Sample volume & repeat testing	Small volume limits repeat measurements and precision assessment.	Larger volumes allow replicate testing, precision evaluation, and multiple comparator methods from one draw.
Practicality for frequent sampling	Repeated fingersticks are impractical and painful during frequent sampling periods.	IV catheter sampling enables high-frequency measurements, especially during clamp studies or hypoglycemia or testing patients who are undergoing hemodialysis and already have an intravenous line in place.
Multi-analyte testing	May be less appropriate for other analytes.	Preferred for simultaneous measurement of multiple analytes (eg, glucose, β-hydroxybutyrate, lactate) and other analytes that relate to interference, such as medications.
Safety during hypoglycemia	Vulnerable to artifactual/pseudohypoglycemia due to peripheral vasoconstriction and reduced capillary flow.	Unaffected by peripheral vasoconstriction, providing more reliable measurements of central circulation in hypoglycemia.
Patient burden & tissue injury	Repeated fingersticks can cause pain, trauma, and scarring.	Indwelling venous lines reduce repeated skin punctures and cumulative injury.

Abbreviations: CGM, continuous glucose monitor; IV, intravenous; MARD, mean absolute relative difference; NIST, national institute of standards and technology.

Advantages of Capillary Blood as a Matrix

The use of capillary blood as a reference matrix has some advantages over venous blood. Historically, CGMs have been calibrated with capillary blood rather than venous plasma. The closer physiological agreement of interstitial fluid glucose measured by a CGM with capillary glucose concentrations is useful for confirming glucose values when self-monitoring of blood glucose is used to make treatment decisions, especially during periods of rapid glucose change. ⁶ These rapid changes can occur after meals (rapid excursions) and sometimes during hypoglycemia, which are times when safety concerns about incorrect insulin management (dosing) are greatest. ¹⁴ The IFCC WG CGM reports rightly praised these features of capillary blood testing. However, a global policy has not been adopted for recommendations regarding a preference for capillary blood over venous blood for reference testing.

Using capillary glucose as the reference for CGM systems may yield a lower mean absolute relative difference (MARD). The lower (better) MARDs seen with capillary blood compared with venous blood are observed if a CGM is calibrated with capillary values, (although CGM companies have been moving away from calibration) and is therefore being evaluated for accuracy against the same matrix as the calibration process. ¹⁵ The apparently better accuracy of capillary-based comparisons is somewhat tautological as it reflects matrix matching between the calibration and reference samples, rather than an intrinsic superiority of capillary blood over venous blood as a physiological compartment or as a biochemical reference standard. CGMs measure glucose in interstitial fluid, which has its own temporal dynamics relative to both capillary and venous blood; there is no inherent reason to consider capillary glucose to be physiologically more accurate than venous glucose. It is worth noting, however, that capillary microsampling can offer practical benefits over venipuncture in certain clinical situations, particularly in pediatric populations. ¹⁶

Advantages of Venous Blood as a Matrix

We believe venous blood is a preferable matrix for comparator reference testing for analytical, practical, and safety reasons. For these reasons, reference testing based on venous blood is widely used in the United States for testing the accuracy of CGM systems.

Analytical Advantages of Venous Blood as Reference Matrix

Venous blood is evaluated on laboratory-grade glucose analyzers. For the United States, these are often Clinical Laboratory Improvement Amendments (CLIA) moderate complexity platforms measuring glucose directly from whole blood or from plasma following centrifugation. In accuracy studies, capillary glucose is often tested on point-of-care blood glucose monitors, which are generally less accurate than laboratory analyzers. Both venous and capillary matrices are best evaluated by centrifuging the blood and testing the plasma on laboratory analyzers. ¹⁷ Traceable, higher-order reference methods are preferred in regulatory submissions, and venous (compared with capillary) samples measured on laboratory analyzers provide a better metrological chain than capillary samples on point-of-care blood glucose monitors. ¹⁸ A YSI or Nova type of benchtop glucose analyzer operates at a higher traceability level and shorter chain involving fewer steps through being calibrated with materials closer to a primary glucose standard. ⁹

For glucose monitors, FDA guidance recommends that the comparator method used in accuracy studies be well validated and traceable to a higher-order reference material and/or method, with the traceability chain described in the submission. In practice, this requirement implies that traceability is established and verified during method validation and study conduct, not retrofitted only after data collection. Mathematical adjustments have been applied retrospectively to reduce or correct bias and align an analyte with NIST-traceable targets tested on an analyzer cleared for testing venous plasma for this purpose. ¹⁹ Such retrospective or post hoc recalibration risks introducing bias and undermining credibility. Post hoc analysis of self-monitoring of blood glucose (SMBG) data cannot be performed because these devices typically do not provide information about their raw signal or calibration algorithms. ²⁰ Preanalytical variability from glycolysis for venous glucose is readily minimized via rapid cooling on ice, rapid testing, and centrifugation. Preanalytical variability is likely higher for capillary versus venous sampling, as incomplete wiping of the first drop, tissue fluid contamination, and variable squeezing are common in capillary testing.

Venous samples compared with capillary samples allow larger volumes and repeat measurements, which support replicate testing, assessment of precision, and evaluation of multiple comparison methods from a single draw, which is difficult with the small volumes of blood extracted for capillary fingerstick sampling. ²¹

Practical Advantages of Venous Blood as Reference Matrix

If frequent sampling is needed during periods of rapid fluctuation or symptomatic hypoglycemia with a clamp study, then venous blood sampling through an intravenous catheter is more practical than repeated fingerstick capillary blood sampling. ²² CGMs are now being designed to measure additional analytes, such as beta hydroxy butyrate and lactate. Analyte concentrations might differ between venous and capillary matrices, ²³ and venous samples are the conventional reference matrix. ²⁴ Consistently measuring glucose in venous blood eliminates the need to test a multianalyte measuring device twice—once with venous blood and once with capillary blood. As new CGMs come on the market measuring glucose in compartments other than in blood or interstitial fluid, such as the intradermal compartment, ²⁵ venous blood will still be a valid comparator, whereas the relationship between capillary glucose and these other compartments might not be as close as between capillary glucose and interstitial fluid glucose.

Safety Advantages of Venous Blood as Reference Matrix

Sympathetic activation during hypoglycemia produces peripheral vasoconstriction and reduced skin/digital blood flow, which can lower capillary glucose and make it harder to obtain an adequate capillary blood sample for reference testing, just when there is the greatest need for an accurate reading. “Artifactual hypoglycemia” or “pseudohypoglycemia” in settings of peripheral vasoconstriction (including Raynaud phenomenon, shock, and severe peripheral vascular disease), where decreased capillary flow and increased local glucose extraction are present can cause spuriously low fingertip glucose readings. ²⁶ Venous blood is unaffected by this process and more reflective of central circulation. Repeated fingersticks can cause cumulative hand trauma, scarring, and pain, whereas an indwelling venous line enables high-frequency sampling without repeated skin punctures. ²⁷

Conclusion

Standards for testing the accuracy of CGMs will likely continue to evolve. One element of a robust protocol for testing glucose in interstitial fluid is selecting a matrix for blood testing. Benefits of comparator testing from venous (compared with capillary) sampling include better traceability, better hypoglycemia characterization, and better analytical sampling with venous testing. Because fingersticks are used as adjunct testing and as long as manufacturer algorithms are designed to align with capillary blood results, capillary testing will always be physiologically aligned with CGM testing. Capillary testing may also be useful during rapid changes in glucose concentrations but is fundamentally limited by analytical rigor and variability during sample collection. Ultimately, as a reference matrix, venous blood offers superior analytical validity, safety, and practicality for CGM accuracy studies.