Capillary Blood Levels of Hydroxychloroquine and Methotrexate Are Stable for up to 5 Years When Collected on Volumetric Absorptive Microsamplers.

BACKGROUND: Hydroxychloroquine (HCQ) and methotrexate (MTX) are common antirheumatic drugs used chronically by patients with systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) (1, 2). Therapeutic drug monitoring (TDM) of HCQ and MTX provides critical compliance information, but typically requires venipuncture and shipment of refrigerated blood samples to the clinical laboratory. Capillary blood collection by finger prick offers a convenient alternative to venipuncture. The long-term stability of capillary blood HCQ and MTX collected using volumetric absorptive microsamplers (VAMS) has not previously been evaluated. In this study, we evaluated the stability of capillary MTX and HCQ levels when stored on VAMS for up to 5 years. METHODS: Samples of patients with RA and SLE were collected for HCQ or MTX monitoring as part of 2 clinical studies in 2015 (HCQ: n = 24 | MTX: n = 61) and 2017 (HCQ: n = 126). Venous and capillary blood samples were shipped to Exagen Inc.'s clinical laboratory. HCQ and MTXPG3 were measured using separate LC-MS/MS methodologies. Baseline venous blood levels of HCQ and MTXPG3 were determined within 1 week of draw and compared to capillary blood levels determined following 3 or 5 years of storage. RESULTS: Venous blood HCQ and capillary blood MTXPG3 determinations made at baseline were significantly different from capillary blood determinations performed following 5 years of storage at -80 °C. However, these differences were within the specified limits of agreement [HCQ: Avg % change after storage (CI 95%) = -7.1% (-12.6, -1.6%), pt-test = 0.0205, interclass correlation coefficient (ICC) = 0.96 | MTXPG3: Avg % change after storage (CI 95%) = 13.3% (7.1, 19.4%), pt-test = 0.0001, ICC = 0.90]. CONCLUSION: Dried capillary blood HCQ and MTXPG3 levels collected on VAMS are stable for up to 5 years.

A Multianalyte Assay Panel With Cell-Bound Complement Activation Products Predicts Transition of Probable Lupus to American College of Rheumatology-Classified Lupus.

OBJECTIVE: To evaluate the usefulness of biomarkers to predict the evolution of patients suspected of systemic lupus erythematosus (SLE), designated as probable SLE (pSLE), into classifiable SLE according to the American College of Rheumatology (ACR) classification criteria. METHODS: Patients suspected of SLE were enrolled by lupus experts if they fulfilled three ACR criteria for SLE and were followed for approximately 1-3 years to evaluate transition into ACR-classifiable SLE. Individual cell-bound complement activation products (CB-CAPs), serum complement proteins (C3 and C4), and autoantibodies were measured by flow cytometry, turbidimetry, and enzyme-linked immunosorbent assay, respectively. Blood levels of hydroxychloroquine (HCQ) were measured by mass spectrometry. A multianalyte assay panel (MAP), which includes CB-CAPs, was also evaluated. A MAP of greater than 0.8 reflected the optimal cutoff for transition to SLE. Time to fulfillment of ACR criteria was evaluated by Kaplan-Meier analysis and Cox proportional hazards model. RESULTS: Of the 92 patients with pSLE enrolled, 74 had one or two follow-up visits 9-35 months after enrollment for a total of 128 follow-up visits. Overall, 28 patients with pSLE (30.4%) transitioned to ACR-classifiable SLE, including 16 (57%) in the first year and 12 (43%) afterwards. A MAP score of greater than 0.8 at enrollment predicted transition to classifiable SLE during the follow-up period (hazard ratio = 2.72; P = 0.012), whereas individual biomarkers or fulfillment of Systemic Lupus International Collaborating Clinics criteria did not. HCQ therapy was not associated with the prevention of transition to SLE. CONCLUSION: Approximately one-third of patients with pSLE transitioned within the study period. MAP of greater than 0.8 predicted disease evolution into classifiable SLE.

Transition of Methotrexate Polyglutamate Drug Monitoring Assay from Venipuncture to Capillary Blood-Based Collection Method in Rheumatic Diseases.

OBJECTIVE: Methotrexate (MTX) polyglutamate (MTXPG3) levels from isolated red blood cells (RBCs) collected by venipuncture have clinical utility in guiding MTX dosing for patients with rheumatoid arthritis (RA). Our objective was to transition this RBC-based therapeutic drug monitoring (TDM) assay to dried capillary blood collected by fingerstick. METHODS: Patients with RA treated with MTX were enrolled. Specimens were collected by fingerstick (volumetric absorptive microsampler) and venipuncture to measure MTXPG3 from dried capillary blood, total venous blood, and isolated RBCs. MTXPG3 levels from dried capillary blood were measured using LC-MS/MS, converted to RBC equivalent (nmol/L), and compared with those from isolated RBCs (reference method). Following transition to fingerstick collection, comparability in the distributions of dried capillary and venipuncture-based RBC MTXPG3 levels was assessed using the Kolmogorov-Smirnov (K-S) test. RESULTS: Intraday and interday precision ranged from 2.0% to 10.9% and 3.1% to 10.8%, respectively, at MTXPG3 concentrations ranging from 5 to 100 nmol/L. In 106 participants treated with MTX, MTXPG3 levels from total venous and dried capillary blood were comparable [slope = 0.97 (95% CI, 0.92-1.03); R 2 = 0.92]. Dried capillary blood MTXPG3 converted to RBC equivalent was similar to levels from isolated RBCs (30 ± 18 nmol/L vs 33 ± 19 nmol/L; n = 106). After implementation in the clinical laboratory, RBC equivalents MTXPG3 from the fingerstick method were similar to levels from venipuncture [39 ± 22 nmol/L (n = 825) vs 39 ± 24 nmol/L (n = 47935)] (K-S test P = 0.09). Underexposure to MTX (MTXPG3 ≤5 nmol/L RBCs) was detected in 7.0% and 8.5% patient specimens collected using the fingerstick and venipuncture methods, respectively. CONCLUSION: Capillary blood MTXPG3 levels can be used to guide MTX dosing in TDM practice.

Capillary blood collected on volumetric absorptive microsampling (VAMS) device for monitoring hydroxychloroquine in rheumatoid arthritis patients.

A novel technique for collection of capillary blood, termed volumetric absorptive microsampling (VAMS), has been recently cleared by the FDA for collection of human blood. VAMS absorbs a fixed volume of blood (10µl) and overcomes area bias and homogeneity issues associated with dried blood spot (DBS). This study is the application of VAMS for therapeutic drug monitoring (TDM) in human capillary blood. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) workflow for analysis of VAMS sample was developed and validated. Concentration of hydroxychloroquine (HCQ) and its metabolites, desethylhydroxychloroquine (DHCQ), desethylchloroquine (DCQ), and bisdesethylchloroquine (BDCQ), in capillary blood on VAMS sampler were compared to those in venous blood in rheumatoid arthritis patients. Feasibility of capillary blood collected on both VAMS and DBS card were evaluated on patients. Stability of dried capillary blood on VAMS was also examined. Our results established that VAMS is a simple and accurate sampling technique that delivers the benefits of DBS sampling while overcoming the issues associated with hematocrit and homogeneity. It requires a small blood volume, simplifies sample logistics management, and may allow sample collection in the patient's home setting.