A sensitive LC-MS/MS methotrexate assay capable of assessing adherence to methotrexate therapy in rheumatoid arthritis.

OBJECTIVES: To develop a sensitive liquid chromatography-tandem mass spectrometry method capable of measuring serum methotrexate in patients with rheumatoid arthritis to assess adherence to drug treatment. METHODS: Isotopically labelled internal standard and deionised water were added to sample prior to solid phase extraction using a Waters Oasis Max ion-exchange 96-well plate. Following extraction, samples were analysed by LC-MS/MS on a TQS-micro mass spectrometer. RESULTS: Mean recovery was 107 % for four different concentrations of methotrexate spiked into seven patient samples, whilst post extraction spiking gave a mean recovery of 100 %. Between-batch and within-batch CVs were ≤6 % at three different concentrations of methotrexate in fresh frozen plasma. Mean bias was <5 % for between-batch and within batch analysis at three different weighed in concentrations of methotrexate certified reference material. The lower limit of quantification of the assay was 0.1 nmol/L with linearity up to approximately 100 nmol/L. Dilution linearity studies were used to validate the dilution of patient samples prior to analysis. There was no significant interference in the method from lipaemia, haemolysis or icterus. CONCLUSIONS: A sensitive LC-MS/MS assay for methotrexate has been developed and validated. The method has been used to measure methotrexate adherence in patient samples from clinical trials and could be used in future research to assess the ability of the assay as a biofeedback intervention to improve adherence to methotrexate therapy.

Diagnostic accuracy of point-of-care tests for detecting albuminuria: a systematic review and meta-analysis.

BACKGROUND: Experts recommend screening for albuminuria in patients at risk for kidney disease. PURPOSE: To systematically review evidence about the diagnostic accuracy of point-of-care (POC) tests for detecting albuminuria in individuals for whom guidelines recommend such detection. DATA SOURCES: Cochrane Library, EMBASE, Medion database, MEDLINE, and Science Citation Index from 1963 through 5 December 2013; hand searches of other relevant journals; and reference lists. STUDY SELECTION: Cross-sectional studies, published in any language, that compared the accuracy of machine-read POC tests of urinary albumin-creatinine ratio with that of laboratory measurement. DATA EXTRACTION: Two independent reviewers extracted study data and assessed study quality using the QUADAS-2 (Quality Assessment of Diagnostic Accuracy Studies 2) tool. DATA SYNTHESIS: Sixteen studies (n = 3356 patients) that evaluated semiquantitative or quantitative POC tests and used random urine samples collected in primary or secondary ambulatory care settings met inclusion criteria. Pooling results from a bivariate random-effects model gave sensitivity and specificity estimates of 76% (95% CI, 63% to 86%) and 93% (CI, 84% to 97%), respectively, for the semiquantitative test. Sensitivity and specificity estimates for the quantitative test were 96% (CI, 78% to 99%) and 98% (CI, 93% to 99%), respectively. The negative likelihood ratios for the semiquantitative and quantitative tests were 0.26 (CI, 0.16 to 0.40) and 0.04 (CI, 0.01 to 0.25), respectively. LIMITATION: Accuracy estimates were based on data from single-sample urine measurement, but guidelines require that diagnosis of albuminuria be based on at least 2 of 3 samples collected in a 6-month period. CONCLUSION: A negative semiquantitative POC test result does not rule out albuminuria, whereas quantitative POC testing meets required performance standards and can be used to rule out albuminuria. PRIMARY FUNDING SOURCE: None.

Investigation of apparent non-albuminuric proteinuria in a primary care population.

BACKGROUND: There is debate as to whether using the urinary albumin- or protein-to-creatinine ratio (ACR or PCR) should be the primary test for proteinuria. Whilst albuminuria (increased ACR) in the absence of proteinuria (increased PCR) may be expected in some patients, the converse (i.e., proteinuria in the absence of albuminuria) is more unusual and its cause and significance are unclear. We investigated the nature of such apparent non-albuminuric proteinuria in a primary care population of patients. METHODS: ACR and PCR were measured in 569 urine samples from patients who either had chronic kidney disease or were at increased risk of the condition. Samples with apparent proteinuria (PCR ≥23 mg/mmol/≥200 mg/g) but no albuminuria (ACR <3.4 mg/mmol/<30 mg/g) were classified as 'discrepant' (37% of proteinuric samples, 6% of all samples); 27 of these samples were available for further analyses. The further analyses included electrophoresis, repeat measurement, immunoassays for markers of tubular proteinuria and use of alternative albumin and total protein methods. RESULTS: Electrophoresis did not identify significant proteinuria in the discrepant samples. The only evidence of tubular proteinuria following measurement of three urinary markers of the condition was a mildly increased α1-microglobulin-to-creatinine ratio in 10 of the 27 discrepant samples analysed, four of which also had a raised ß-trace protein-to-creatinine ratio. Use of an alternative urinary total protein method resulted in significantly lower PCRs and 17 of the 27 samples were no longer classified as proteinuric. CONCLUSIONS: We were unable to confirm the cause of a raised PCR without albuminuria in these patients and suspect that in most cases it is artefactual.

The diagnostic accuracy of a urine albumin-creatinine ratio point-of-care test for detection of albuminuria in primary care.

BACKGROUND: Albuminuria is an important sign of chronic kidney disease and is detected routinely by measurement of urinary albumin-creatinine ratio (ACR). A Siemens CLINITEK test designed for use at the point of care is available that can semiquantitatively measure ACR. STUDY DESIGN: Diagnostic accuracy study evaluating a urinary ACR point-of-care test. SETTING & PARTICIPANTS: The semiquantitative ACR test was evaluated at the point of care in a representative primary care population (those with or at increased risk of chronic kidney disease) of 642 patients under standard operational conditions and compared with the reference standard of ACR measurement in the clinical laboratory. INDEX TEST: The point-of-care CLINITEK semiquantitative ACR test. This test uses dye-binding and catalytic assays for albumin and creatinine, respectively, on a Microalbumin 9 strip, which is read by the CLINITEK Status Analyzer, and ACR is calculated automatically. REFERENCE TEST: Laboratory measurement of albumin and creatinine on an Abbott Architect analyzer by immunoturbidimetric and enzymatic assays, respectively, and calculation of ACR. RESULTS: The prevalence of albuminuria (laboratory ACR≥30 mg/g) in the study population was 20.2%. Sensitivity and specificity of the point-of-care test for detecting albuminuria were 83.2% and 80.0%, respectively. Positive and negative predictive values were 51.2% and 95.0%, respectively; positive and negative likelihood ratios were 4.16 and 0.21, respectively. Twenty-three (3.6%) samples measured at the point of care were not analyzed in the central laboratory for a variety of reasons, including laboratory reception data entry errors. LIMITATIONS: Our sensitivity calculation is accurate to an approximately 8% CI. CONCLUSIONS: The instrument-read reagent strip test was a poor rule-in test for albuminuria at the point of care, as evidenced by the low positive predictive value, but was a reasonable rule-out test. Observed sensitivity was lower than reported in earlier laboratory-based studies. This decreased diagnostic accuracy needs to be balanced against the potential advantages of a point-of-care testing approach.

A rapid LC-MS/MS assay for the measurement of serum methotrexate in patients who have received high doses for chemotherapy.

BACKGROUND: Methotrexate is used in high doses to treat a number of cancers, particularly certain haematological malignancies. Monitoring of serum methotrexate concentration is important due to the potential toxicity of methotrexate and the variation in methotrexate pharmacokinetics in different patients on the same treatment regimen. OBJECTIVE: To develop a rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for monitoring serum methotrexate in patients on high-dose chemotherapy. METHOD: Isotopically labelled internal standard was added to sample prior to protein precipitation with methanol. Diluted supernatant was injected into a Waters Acquity UPLC system linked to a TQS-Micro mass spectrometer. Separation by chromatography was achieved with a Waters Phenyl Vanguard with a retention time of approximately 0.5 min. The quantifier and qualifier transitions for methotrexate were 455.2>134.1 and 455.2>175.2, respectively. RESULTS: Mean recovery was 111% for three different concentrations of methotrexate spiked into seven different patient samples, with ion suppression <1%. Between-batch and within-batch coefficient of variations were <5% at three different concentrations of methotrexate in fresh frozen plasma. The lower limit of quantification was 0.02 µmol/L and the assay was shown to be linear to approximately 25 µmol/L. The LC-MS/MS assay showed a mean bias of -8.6% compared to an immunoassay, while mean bias compared to weighed in targets in external quality assessment samples was 1.6%. DISCUSSION: A rapid LC-MS/MS assay for methotrexate has been developed and validated. The LC-MS/MS method is likely to offer superior accuracy and specificity to more widely available immunoassays.

Replacing urine protein electrophoresis with serum free light chain analysis as a first-line test for detecting plasma cell disorders offers increased diagnostic accuracy and potential health benefit to patients.

OBJECTIVES: To determine the most clinically effective diagnostic testing strategy for plasma cell disorders in the clinical laboratory. METHODS: Serum and urine samples from 2,799 patients with suspected plasma cell dyscrasias were tested by alternative diagnostic testing strategies consisting of serum protein electrophoresis (SPE) with either urine protein electrophoresis (UPE) or serum free light chain (sFLC) analysis. RESULTS: The combination of sFLC analysis and SPE had the greatest sensitivity (100%), detecting abnormalities in all 124 patients diagnosed with plasma cell disorders. Routine sFLC testing would have had much potential health benefit for two patients in the study population. First, a patient who had a markedly abnormal sFLC result was diagnosed with light chain deposition disease by renal biopsy, but no abnormality was detected by SPE or UPE. Second, a patient diagnosed with multiple plasmacytomas following biopsy of a lung tumor had a grossly abnormal sFLC result but an equivocal weak-positive SPE result, and no urine sample was received by the laboratory for the patient. CONCLUSIONS: Our study suggests that the combination of SPE and sFLC analysis is the most clinically effective first-line diagnostic testing strategy for detecting plasma cell disorders in the clinical laboratory.

Identification of a macro-alkaline phosphatase complex in a patient with inflammatory bowel disease.

We report the rare finding of a macro-alkaline phosphatase (macroALP) complex in a patient with a previously unexplained raised alkaline phosphatase activity. The clinical symptoms were persistent, daily diarrhoea for two months with blood in the stool. The patient was subsequently diagnosed with inflammatory bowel disease, specifically ulcerative colitis, following a rectal biopsy and colonoscopy. Two cases of macroALP associated with ulcerative colitis have been reported before, suggesting there could be an increased prevalence of macroALP in these patients.