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Validation of a flash glucose monitoring system in outpatient diabetic cats

Abstract Background Interstitial glucose (IG) concentration measurement using a flash glucose monitoring system (FGMS) is a noninvasive, affordable, and informative method to regulate patients with diabetes mellitus (DM) but has not been fully validated in outpatient cats with DM. Objectives To further validate the FreeStyle Libre FGMS in outpatient diabetic cats. Animals Eight client‐owned cats with DM. Methods Prospective observational validation study. Tissue glue was used to attach the sensor to the cat. Lin's concordance correlation coefficient (ρ c) was used to compare IG concentrations measured by the FGMS to blood glucose concentrations measured using an automated biochemistry analyzer (ABA) and point‐of‐care glucometer (POCG). Results Data from 15 sensor placements in 8 cats were analyzed. Paired IG and ABA glucose concentrations (139 samples) had excellent correlation (ρ c = 0.96) as did IG and POCG glucose concentrations (142 samples, ρ c = 0.92). Sensor failure or displacement were recorded for 12/15 (80%) sensor placements. Median time of sensor activity was 7 days (range, 2‐13 days). Conclusions and Clinical Importance In outpatient cats with DM, the FGMS‐measured IG concentration correlated well with ABA‐measured blood glucose concentration, but a high rate of sensor failures was observed.

serial blood sampling and can provide a reflection of the average glucose concentration over the preceding 1 to 2 weeks. [7][8][9] However, serum fructosamine concentration does not provide information about fluctuations in blood glucose concentration and also is affected by serum albumin and thyroid hormone concentrations. 10 Continuous glucose monitoring devices are less invasive than serial venipuncture and provide substantially more data regarding glucose concentration fluctuations over time. Various continuous glucose monitoring systems previously have been investigated for use in diabetic cats, but the clinical use of these devices is limited because they are physically cumbersome, require at least once daily calibration with blood glucose concentration, and are expensive. [11][12][13][14][15][16][17] A newer flash glucose monitoring system (FGMS; FreeStyle Libre, Abbott, Alameda, California) is appealing for use in cats because of its small size, ease of use, affordability, intuitive computer software, and factory calibration which eliminates the need for venipuncture and calibration with blood glucose concentration. The FGMS has been validated in dogs with DM treated on an outpatient basis as well as in dogs hospitalized for diabetic ketoacidosis. [18][19][20][21][22] One study also has validated the FGMS in hospitalized diabetic cats. 23 In this study, the investigators used 8 sutures to attach the sensor to mostly nonsedated cats, a technique that could be unacceptable to some veterinarians and owners. 23 Another recent study described complications associated with the FGMS in diabetic cats, highlighting the commonplace use of this device and the need for further validation of its accuracy. 24 Our primary aim was to validate the use of the FGMS in outpatient diabetic cats using tissue glue to attach the sensor to the cat. A secondary aim was to assess the tolerability and adverse events associated with the sensor in cats.

| Study population
Client-owned cats with naturally occurring DM that were examined at a university teaching hospital between April 2019 and July 2020 were prospectively enrolled in an observational validation study. Inclusion criteria included a diagnosis of DM, a body weight >3 kg, a body condition score ≥4/9, and a state of hydration (≤5% dehydrated). Owners also had to consent to three 10-hour outpatient visits within the 13-day period. Exclusion criteria included anemia (PCV < 27%) on the day of enrollment and recent exposure to drugs that can interfere with FGMS readings (acetaminophen, dopamine, icodextrin, salicylates, and ascorbic acid). 25 Additionally, cats could not undergo anesthesia, radiography, computed tomography, or magnetic resonance imaging for the duration of the 13-day study period, because the FGMS sensor can be affected by these procedures. 25 Cats were included regardless of their insulin treatment regimen, which was dictated by the attending clinician. Cats with concurrent diseases and receiving various other medications were included. The University Privately Owned Animal Protocol Committee and the Institutional Animal Care and Use Committee approved the study and informed consent form, and owners signed the written informed consent form before enrollment.

| Data collection
On the day of enrollment (day 0), a 14-day FGMS sensor was placed in a standardized fashion by 1 of the authors (E.K. Shea) as previously described. 21 Briefly, an approximately 5 Â 5 cm square area of skin on the dorsal neck was clipped and cleaned with dilute chlorhexidine followed by alcohol, and the area was allowed to dry fully for at least 1 minute. The sensor was placed according to the manufacturer's guidelines, with the addition of 3 to 4 drops of tissue glue on the adherent side of the sensor. 26 The sensor applicator was held firmly in place for 30 seconds after deployment of the sensor. No other adhesives or bandages were used, but some owners chose to place a stockinette on their cat to prevent removal of the sensor. Cats were fed and received their insulin and any other medications at home, before arrival at the hospital.
Data collection also was performed as previously described. 21 For each cat on day 0, the first blood sample was collected after a 1-hour FGMS calibration period. A maximum of 2 mL was obtained from a peripheral or jugular vein for measurement of PCV, total protein concentration, point-of-care blood glucometer reading (POCG; Accu-Chek Performa, Roche Diagnostics Corp, Indianapolis, Indiana), and blood glucose concentration measurement on the automated biochemistry analyzer (ABA; Vitros 4600 Chemistry System, Ortho-Clinical Diagnostics, Rochester, New York). Subsequent blood samples were collected at 2-hour intervals for a total of 5 samples per day per cat, for measurements of blood glucose concentration using the POCG and ABA. These 5 blood sample measurements at 2-hour intervals also were performed during 2 follow-up visits at approximately 1-week intervals. Interstitial glucose (IG) concentration was measured by scanning the FGMS sensor using the cat's designated reader within 1 minute of blood collection. The POCG glucose concentration measurements were performed using 1 drop of whole blood immediately after venipuncture while ABA blood glucose concentration measurements were performed on serum. A single, study-designated, POCG device was used throughout the study. For ABA glucose concentration measurements, blood samples were submitted immediately to the in-house clinical pathology laboratory and centrifuged within 15 minutes of venipuncture. The ABA utilized in this study uses a colorimetric glucose oxidase method to quantify serum glucose concentration. The serum of each sample also was analyzed for evidence of hemolysis, lipemia, and icterus by trained laboratory technicians and subjectively graded as none, mild, moderate, or marked.
The sensor was left in place for the 13-day study period, and owners were instructed to scan the sensor with the reader at least 3 times per day at 8-hour intervals, because the sensor can store data for a maximum of 8 hours. Each cat returned for 2 follow-up visits at approximately 1-week intervals. During these visits, the same procedure as on the day of enrollment was followed, but the 1-hour calibration period was not necessary because the sensor was already in place. If the sensor fell off or was removed before the intended 13-day study duration, it was replaced by 1 of the authors (E.K. Shea) and the same protocol as for the day of enrollment was followed. At each of the visits, owners completed a standardized questionnaire about their cat's clinical signs, medications, times of meals and insulin administration, and any FGMS sensor-related concerns. At the end of the final visit day, the sensor was removed by 1 of the authors (E.K. Shea).

| Validation data analysis
All samples were analyzed together and in subgroups based on the ABA glucose concentration. Hypoglycemia, normoglycemia, hyperglycemia, and pronounced hyperglycemia were defined as an ABA measured blood glucose concentration of <67, 67-168, >168, and >250 mg/dL, respectively. The detection limits of the FGMS sensor and POCG are 40 to 500 mg/dL and 10 to 600 mg/dL, respectively, and results outside of the detection limits were excluded from the validation analysis. Erroneous FGMS results and samples with insufficient volume also were excluded. For hemolysis, lipemia, and icterus, samples graded as "none" or "mild" were grouped together, and those graded as "moderate" or "marked" were grouped together.

| Sample size calculation
Three sample size calculations were performed for the validation study. Two-sided paired t tests were used to determine the number of samples required to detect a difference of at least 15 mg/dL between blood glucose concentration measured by the ABA and IG concentration measured by the FGMS. A difference of 15 mg/dL was chosen as the smallest clinically important difference that would be meaningful to detect. The calculation for samples with normoglycemia was made on the basis of the mean ± SD glucose concentration (99 ± 13 mg/dL) used to establish the reference interval for the ABA.
The calculation for samples with hyperglycemia was made using a mean of 250 ± 13 mg/dL and the calculation for samples with hypoglycemia was made using a mean of 67 ± 13 mg/dL. It was assumed that the SD of glucose concentrations determined by the FGMS would be similar to that of the glucose concentrations determined by the ABA and that the SD of glucose concentration with hypoglycemia and hyperglycemia would be the same. Additional assumptions included a power of 0.8, type I error rate of 0.05, and a ratio of 1 between the ABA and FGMS sample sizes. The calculation resulted in a required sample size of 13 ABA and FGMS paired measurements for each of the 3 categories of glucose concentration for a total of 39 paired measurements. Each cat contributed 15 paired glucose measurements by providing 5 blood glucose and IG concentration measurements per day on 3 separate days over the 13-day study period. Therefore, a minimum of 3 cats were required for the study.
However, in dogs, approximately 30% of sensors were reported to fall out unintentionally, and preliminary personal experience of the investigators with the device indicated that the attrition rate in cats could be higher, and thus 15 sensor placements were included in the study. 18,21

| Statistical analysis
Variables were assessed for normality visually and using skewness/kurtosis tests for normality. Most variables, including ABA, POCG, and FGMS glucose concentrations, were not normally distributed and therefore are reported as median (range) or count and percentage.
Lin's concordance correlation coefficient (ρ c ) was used to compare the gold standard ABA to FGMS and POCG glucose concentrations, as previously reported. 21 Bland-Altman plots were generated to visually evaluate the correlations. A multivariable linear regression model was used to evaluate the effects of ABA, PCV, TP, hemolysis, lipemia, and icterus on FGMS glucose concentration measurements.
Two mixed effects models were fitted to the data to account for glucose variability among cats, as previously reported in dogs. 21 Interstitial glucose concentration was modeled as a function of blood glucose concentration as follows: with β 0 + β 1* (ABA measured blood glucose concentration) serving as the fixed effects of the model, the random effect occurring at the cat level, and an error term that is a function of undetermined factors such as within cat variability, sleep time, or mealtime. For the purpose of comparison, POCG glucose concentration measurements were similarly modeled as a function of blood glucose concentration as follows: IG concentration ¼ β 0 þ β 1* ABA measured blood glucose concentration ð Þ þ random effect on cat þ error term, POCG glucose concentration ¼ β 0 þ β 1* ABA measured blood glucose concentration ð Þ þ random effect on cat þ error term: The normality of the residuals of the generalized linear models was confirmed graphically by plotting kernel density estimates and standardized normal probability (P-P) plots. P-values <.05 were con- Ohio), ursodeoxycholic acid (compounded formulation), ocular medications, and fiber and probiotic supplementation.
One cat was assessed subjectively to be mildly dehydrated (<5%) on the first day of the study but this cat was assessed to be normally hydrated on the remaining days. All other cats were assessed to be subjectively well-hydrated on examination on all 3 in-hospital days.

| Validation data
Descriptive statistics for paired glucose concentrations measured by the FGMS, POCG, and ABA are presented in Table 1 was significantly different from 0 (P < .001). All other correlation coefficients in the different subgroups of glucose concentrations were significantly different from 0 (P < .001 for all). A Bland-Altman limitsof-agreement plot demonstrating these correlations is presented in Figure 1, and a Lin's concordance correlation graph is presented in Figure 2.
Results of the mixed effect models are presented in Table 3 and indicate that a significant positive correlation existed between the FGMS and ABA glucose concentrations as well as between POCG and ABA glucose concentrations (P < .001 for both). These models predict that for every 1 mg/dL increase in ABA measured blood glucose concentration, there is an increase in 0.97 mg/dL on the FGMS measured IG concentration and an increase in 0.94 mg/dL on the POCG measured blood glucose concentration. Both mixed effect models differed significantly from the ordinary linear regression models indicating that the random effects were significant in both models.
T A B L E 2 Lin's concordance correlation analyses of the differences among the gold standard automated biochemistry analyzer serum glucose concentration, interstitial glucose concentration measured by the flash glucose monitoring system, and blood glucose concentration measured by the point-of-care glucometer

| Sensor tolerability and adverse events
In all cats, the sensor was placed on the dorsal neck in a standardized F I G U R E 2 Lin's concordance correlation graphs of the gold standard automated biochemistry analyzer serum glucose concentration compared to interstitial glucose concentration measured by the flash glucose monitoring system (A), blood glucose concentration measured by the point-of-care glucometer compared to interstitial glucose concentration measured by the flash glucose monitoring system (B), and blood glucose concentration measured by the point-of-care glucometer compared to the gold standard automated biochemistry analyzer serum glucose concentration (C) in all samples. For each graph, the green line represents the reduced major axis and the red line represents the line of perfect concordance. The closer the green line is to the red line, the better the correlation between the 2 compared glucose concentrations T A B L E 3 Results of mixed effects models modeled as: Interstitial glucose concentration measurements = β 0 + β 1* (ABA measured blood glucose concentration) + random effect on cat + error term and point-of-care glucometer glucose concentration measurements = β 0 + β 1* (ABA measured blood glucose concentration) + random effect on cat + error term nor was it stated whether cats exposed to drugs that could influence sensor function were included in the study. 25 Our results are similar to those of previous studies validating the   24 The authors of that study postulated that the use of cyanoacrylate tissue glue combined with the sensor adhesive may have contributed to the development of more severe skin reactions, and tissue glue therefore was not used in the majority of the cats in that study. 24 Interestingly, the same tissue glue utilized in that study was used for all cats in our study and only minor skin reactions were noted in 2/15 sensor placements. Only 3 to 4 drops of glue were used for sensor placement in our study, and thus the amount of glue utilized or other patient-specific factors could have contributed to this difference. The higher rate of sensor detachment in our study could be a result of the location of sensor placement, which was the dorsal neck in our study compared to primarily the dorsolateral thorax in the previous study. 24 Another larger study of the same FGMS in hospitalized cats found a similar complication rate to the current study, with 70% of sensors failing after a median of 8.3 days despite fixing the sensor to the cat with 8 sutures. 23 Based on our results and the 2 previously described studies in cats, FGMS sensor failure appears to be more common in cats compared to dogs, in which sensor failure rates range from 30% to 44% in outpatients. 18,21 This higher rate of sensor failure in Cats included in the study generally had poorly controlled DM as well as a variety of concurrent diseases and were on various noninsulin medications, which could have affected sensor application and tolerability, but this study population reflects the clinical population of diabetic cats at a tertiary referral hospital, and allows for generalizability of the findings to the true population of cats with DM.
In conclusion, we further validated the FGMS for use in outpatient cats with DM using tissue glue to adhere the sensor to the cat, but a higher rate of sensor detachment is expected in cats compared to dogs. Additional studies examining the optimal method and location of sensor application as well as validating the use of the device in the context of diabetic crises and hypoglycemia are needed.

ACKNOWLEDGMENT
The study was made possible by a gift from Ms. Catharine Adler.

CONFLICT OF INTEREST DECLARATION
Authors declare no conflict of interest.

OFF-LABEL ANTIMICROBIAL DECLARATION
Authors declare no off-label use of antimicrobials.

INSTITUTIONAL ANIMAL CARE AND USE COMMITTEE (IACUC) OR OTHER APPROVAL DECLARATION
Approved by the University of Pennsylvania IACUC, protocol number 806686.

HUMAN ETHICS APPROVAL DECLARATION
Authors declare human ethics approval was not needed for this study.