A-117 An intriguing case of the green plasma

Abstract Background A green-colored plasma sample sent to the lab for chemistry testing raises concerns about potential interferences. After consultation with the clinical care team, the source of the green coloration is determined to be methylene blue. Methylene blue is commonly used in the treatment of methemoglobinemia but can also be administered as a surgical dye. In this case, methylene blue is administered to a breast cancer patient for sentinel node mapping. Methylene blue exhibits peak light absorption at 292 nm, 613 nm, and 664 nm, and the reduction of methylene blue to leucomethylene blue, facilitated by methemoglobin reductase, may potentially cause chemical interference by affecting NADPH oxidation. Therefore, the impact of methylene blue on several spectrophotometric assay and electrochemiluminescence assays measure on the Roche Cobas analyzer was investigated Methods The investigation consisted of two arms: review of documents and review of reaction monitor curves. The Elecsys Troponin T hs assay, a heterogeneous sandwich assay using electrochemiluminescence, is performed on the Cobas® e801 module. The Creatine Kinase assay on the Cobas® c702 module involves a two-step process where creatine kinase activity is coupled with the glucose-hexokinase reaction, converting NADP+ to NADPH, which is measured spectrophotometrically at 340 nm. The hexokinase and G6PDH enzymes, and ADP and NADP+ substrates are first added to react with endogenous creatine phosphate, ATP and glucose molecules to produce a baseline absorbance. Excess creatine phosphate and glucose is then added in to initiate the enzymatic reaction for CK determination. The Glucose HK assay Gen.3 (NADPH, 340 nm), Iron Gen. 2 (Fe2+: FerroZine complex, 570 nm), Calcium Gen. 2 (Ca2+: 5-nitro 5’ methyl BAPTA complex, 340 nm), and Magnesium Gen. 2 (Mg2+: xylidyl blue complex, 600 nm) assays were also performed to study spectral and chemical interferences from methylene blue. Controls were run using QC and samples from other patients Results Methylene blue is not listed as a potential interference in the package insert for the tests performed, and no interference has been reported in the literature. Both the e801 and c702 module on the Cobas® analyzer did not raise any alarms or display any error messages such as abnormal absorbance when the sample is analyzed. No reaction monitoring curve was obtained for the Elecsys Troponin T hs assay due to the assay*s design. The baseline CK signal for the sample is lower compared to the controls, but the reaction profiles are the same for samples and controls at baseline and after the addition of the second reagent. The baseline signals for glucose and calcium in the sample are slightly lower than those the controls, while the baseline signals for iron and magnesium are higher. However, the profile of the reaction monitoring curve are the same between the patient and controls. Conclusion Evaluation of the package insert, review of literature, and analysis of reaction tracing suggest methylene blue causes minimal interference. However, given that a formal spiking study was not conducted to confirm no interference was present, the released results were appended with