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.

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.

Detection of anti-dsDNA antibodies by computer-aided automated immunofluorescence analysis.

INTRODUCTION: NOVA View is a computer aided fluorescence microscope that is used for the automated reading and interpretation of indirect immunofluorescent tests in diagnostic immunology. The objective of the present study was to evaluate the performance of the NOVA View® system for the measurement of anti-dsDNA antibodies using the Crithidia luciliae indirect immunofluorescence test (CLIFT) technology. METHODS: Analytical performance of NOVA View CLIFT was assessed in repeatability (within run) and reproducibility (between runs and instruments) studies. Two hundred-fifty patient samples (N=200 consecutive samples and N=50 samples from systemic lupus erythematosus patients) were tested to evaluate the agreement between results generated with NOVA View CLIFT, and those obtained with manual microscopic reading of the same slides. Positivity rate in SLE was assessed on the 50 SLE samples. RESULTS: The NOVA View system showed high level of repeatability and reproducibility within runs, between runs, and between instruments. Agreement of NOVA View software interpretation and digital image reading results with manual microscopic reading results was 96.0%, and the same positivity rate was obtained on SLE samples by NOVA View digital image reading as that of manual microscopic reading (36.0% vs. 38.0%, respectively). CONCLUSION: Results generated by NOVA View CLIFT were equivalent to those obtained by manual microscopic reading on a large routine sample set. NOVA View demonstrated consistency within and between runs, and between instruments. Automation of CLIFT provides reliability and is a suitable alternative for routine clinical laboratories.