A protocol for testing the stability of biochemical analytes. Technical document.

Stability of a measurand in a specimen is a function of the property variation over time in specific storage conditions, which can be expressed as a stability equation, and is usually simplified to stability limits (SLs). Stability studies show differences or even inconsistent results due to the lack of standardized experimental designs and heterogeneity of the chosen specifications. Although guidelines for the validation of sample collection tubes have been published recently, the measurand stability evaluation is not addressed. This document provides an easy guideline for the development of a stability test protocol based on a two-step process. A preliminary test is proposed to evaluate the stability under laboratory habitual conditions. The loss of stability is assessed by comparing measurement values of two samples obtained from the same patient and analyzed at different time points. One of them is analyzed under optimal conditions (basal sample). The other is stored under specific stability conditions for a time set by the laboratory (test sample). Differences are expressed using percentage deviation (PD%) to facilitate comparison with specifications. When the preliminary test demonstrates instability, a comprehensive test is proposed in order to define the stability equation and to specify SLs. Several samples are collected from a set of patients. The basal sample is analyzed under optimal conditions, whereas analysis of test samples is delayed at time intervals. For each patient PD% is calculated as the difference between measurements for every test sample and its basal one and represented in a coordinate graph versus time.

Laboratory sample stability. Is it possible to define a consensus stability function? An example of five blood magnitudes.

BACKGROUND: The stability limit of an analyte in a biological sample can be defined as the time required until a measured property acquires a bias higher than a defined specification. Many studies assessing stability and presenting recommendations of stability limits are available, but differences among them are frequent. The aim of this study was to classify and to grade a set of bibliographic studies on the stability of five common blood measurands and subsequently generate a consensus stability function. METHODS: First, a bibliographic search was made for stability studies for five analytes in blood: alanine aminotransferase (ALT), glucose, phosphorus, potassium and prostate specific antigen (PSA). The quality of every study was evaluated using an in-house grading tool. Second, the different conditions of stability were uniformly defined and the percent deviation (PD%) over time for each analyte and condition were scattered while unifying studies with similar conditions. RESULTS: From the 37 articles considered as valid, up to 130 experiments were evaluated and 629 PD% data were included (106 for ALT, 180 for glucose, 113 for phosphorus, 145 for potassium and 85 for PSA). Consensus stability equations were established for glucose, potassium, phosphorus and PSA, but not for ALT. CONCLUSIONS: Time is the main variable affecting stability in medical laboratory samples. Bibliographic studies differ in recommedations of stability limits mainly because of different specifications for maximum allowable error. Definition of a consensus stability function in specific conditions can help laboratories define stability limits using their own quality specifications.

Long-term metabolic correction of Wilson's disease in a murine model by gene therapy.

BACKGROUND & AIMS: Wilson's disease (WD) is an autosomal recessively inherited copper storage disorder due to mutations in the ATP7B gene that causes hepatic and neurologic symptoms. Current treatments are based on lifelong copper chelating drugs and zinc salts, which may cause side effects and do not restore normal copper metabolism. In this work we assessed the efficacy of gene therapy to treat this condition. METHODS: We transduced the liver of the Atp7b(-/-) WD mouse model with an adeno-associated vector serotype 8 (AAV8) encoding the human ATP7B cDNA placed under the control of the liver-specific α1-antitrypsin promoter (AAV8-AAT-ATP7B). After vector administration we carried out periodic evaluation of parameters associated with copper metabolism and disease progression. The animals were sacrificed 6months after treatment to analyze copper storage and hepatic histology. RESULTS: We observed a dose-dependent therapeutic effect of AAV8-AAT-ATP7B manifested by the reduction of serum transaminases and urinary copper excretion, normalization of serum holoceruloplasmin, and restoration of physiological biliary copper excretion in response to copper overload. The liver of treated animals showed normalization of copper content and absence of histological alterations. CONCLUSIONS: Our data demonstrate that AAV8-AAT-ATP7B-mediated gene therapy provides long-term correction of copper metabolism in a clinically relevant animal model of WD providing support for future translational studies.

Analysis of common biomarkers in capillary blood in routine clinical laboratory. Preanalytical and analytical comparison with venous blood.

OBJECTIVES: Remote self-collected capillary blood samples have been proposed as alternative to venous blood samples as an aid in telemedicine. The aim of this work is to compare the preanalytical and analytical performance of these two types of samples and to study the stability of common measurands in capillary blood. METHODS: Capillary and venous blood samples were collected in parallel from 296 patients in serum tubes to analyze 22 common biochemistry magnitudes after centrifugation and in EDTA tubes to analyze 15 hematologic magnitudes. Quality of the preanalytical process was assessed applying the model of quality indicator. 24 h stability at room temperature was studied by obtaining paired capillary samples. A questionnaire of assessment was conducted. RESULTS: Mean hemolysis index was higher in capillary samples compared to venous blood samples (p<0.001). Regression analysis and difference analysis showed no bias for all studied biochemistry parameters and hematologic parameters, except mean corpuscular volume (MCV), between capillary and venous blood samples. Regarding sample stability, percentage deviation was higher than the corresponding minimum analytical performance specification for ferritin, vitamin D, hematocrit, MCV, mean corpuscular hemoglobin concentration, platelets distribution wide, mean platelet volume and basophils. Finger pricking was perceived as less painful (p<0.05) than venipuncture in participants who undergo more than one blood test per year. CONCLUSIONS: Capillary blood can be used as an alternative to venous blood for the studied parameters in automated common clinical analyzers. Cautious should be taken if samples are not analyzed within 24 h from the collection.

Influence of study model, baseline catalytic concentrations and analytical system on the stability of serum alanine aminotransferase.

Objectives: The stability of the analytes most commonly used in routine clinical practice has been the subject of intensive research, with varying and even conflicting results. Such is the case of alanine aminotransferase (ALT). The purpose of this study was to determine the stability of serum ALT according to different variables. Methods: A multicentric study was conducted in eight laboratories using serum samples with known initial catalytic concentrations of ALT within four different ranges, namely: <50 U/L (<0.83 µkat/L), 50-200 U/L (0.83-3.33 µkat/L), 200-400 U/L (3.33-6.67 µkat/L) and >400 U/L (>6.67 µkat/L). Samples were stored for seven days at two different temperatures using four experimental models and four laboratory analytical platforms. The respective stability equations were calculated by linear regression. A multivariate model was used to assess the influence of different variables. Results: Catalytic concentrations of ALT decreased gradually over time. Temperature (-4%/day at room temperature vs. -1%/day under refrigeration) and the analytical platform had a significant impact, with Architect (Abbott) showing the greatest instability. Initial catalytic concentrations of ALT only had a slight impact on stability, whereas the experimental model had no impact at all. Conclusions: The constant decrease in serum ALT is reduced when refrigerated. Scarcely studied variables were found to have a significant impact on ALT stability. This observation, added to a considerable inter-individual variability, makes larger studies necessary for the definition of stability equations.