Ammonia is an end-product of deamination of amino acids. In vivo, it is physiologically produced, predominantly in the gastrointestinal tract, myocytes, lymphocytes, and the kidney. Ammonia is neurotoxic if it accumulates but, normally, it is metabolized to urea by hepatocytes, and to glutamine by myocytes and hepatocytes. Urea is excreted by the kidney, but can also diffuse into all tissues and body fluids. In the gastrointestinal tract, bacterial ureases produce ammonia from the urea. Significant formation of ammonia also occurs following hydrolysis of glutamine by glutaminase containing cells such as gastrointestinal epithelial cells and lymphocytes [1].
Evaluation of plasmatic ammonia is a prognostic test primarily performed in the clinical …show more content…
In particular, pre-analytical conditions, which may lead to overestimated results. For example, delayed sample processing, environmental contamination following cigarette smoking or use of ammonia-containing detergents are some of the factors that cause pre-analytical errors. For these reasons, the existing literature [1,7] and ammonia assay manufacturers recommend: a) use ethylenediaminetetraacetic acid (EDTA) as a preservative, since heparin interferes in the ammonia assay; b) avoid serum, because during the coagulation process ammonia could be produced; c) collect the blood from the veins without stasis; d) prohibit smoking to the patient before sampling; e) after collection, samples should be immediately put in ice and, after centrifugation at 4°C, the test should be performed within 20-30 minutes of …show more content…
Such interferences in patient specimens increase the background absorbance in the reaction which is not alleviated by subtracting the secondary absorbance reading at 700 nm. For the Cobas c501 module, this results in an increased reagent background absorbance to a level which exceeds the maximum absorbance specified for the instrument detector (32000 ABS), once reagent 3 (NADPH) is added to the reaction (Figure 1).
In 2014, Roche released a technical bulletin [8], advising customers to allow the reagent rest open on the shelf for 24 h to release ammonia eventually built up, prior to loading on the instruments. Our laboratory adopted this process without significant effects on reducing instrumental error flag rates.
In this paper, we present a variation developed by our research group, of the Roche NH3L method, with the aim of significantly reducing this kind of alarm. We proposed a different dilution of the reagent 3 in order to reduce, in terms of absorbance, the signal generated by the reaction itself. Following several tests, a decrease of error flag "> ABS" equal to 95% on the c501 module has been observed. This method, named “NH3L open” has been validated in terms of imprecision by within-run and between-run tests, and subsequently correlated to the classic method installed on Cobas C6000 c501
Evaluation of plasmatic ammonia is a prognostic test primarily performed in the clinical …show more content…
In particular, pre-analytical conditions, which may lead to overestimated results. For example, delayed sample processing, environmental contamination following cigarette smoking or use of ammonia-containing detergents are some of the factors that cause pre-analytical errors. For these reasons, the existing literature [1,7] and ammonia assay manufacturers recommend: a) use ethylenediaminetetraacetic acid (EDTA) as a preservative, since heparin interferes in the ammonia assay; b) avoid serum, because during the coagulation process ammonia could be produced; c) collect the blood from the veins without stasis; d) prohibit smoking to the patient before sampling; e) after collection, samples should be immediately put in ice and, after centrifugation at 4°C, the test should be performed within 20-30 minutes of …show more content…
Such interferences in patient specimens increase the background absorbance in the reaction which is not alleviated by subtracting the secondary absorbance reading at 700 nm. For the Cobas c501 module, this results in an increased reagent background absorbance to a level which exceeds the maximum absorbance specified for the instrument detector (32000 ABS), once reagent 3 (NADPH) is added to the reaction (Figure 1).
In 2014, Roche released a technical bulletin [8], advising customers to allow the reagent rest open on the shelf for 24 h to release ammonia eventually built up, prior to loading on the instruments. Our laboratory adopted this process without significant effects on reducing instrumental error flag rates.
In this paper, we present a variation developed by our research group, of the Roche NH3L method, with the aim of significantly reducing this kind of alarm. We proposed a different dilution of the reagent 3 in order to reduce, in terms of absorbance, the signal generated by the reaction itself. Following several tests, a decrease of error flag "> ABS" equal to 95% on the c501 module has been observed. This method, named “NH3L open” has been validated in terms of imprecision by within-run and between-run tests, and subsequently correlated to the classic method installed on Cobas C6000 c501