PEPFAR Quality Control and Method Validation Activity 14 Handout 1: AST

Handout 1: AST 1403 ASTL Aspartate Aminotransferase acc. to IFCC without pyridoxal phosphate activation Intended use Limitations - interferences In vitro test for the quantitative determination of aspartate aminotransferase (AST) in human serum and plasma Criterion: Recovery within ± 10 % of initial value at an AST activity of 30 U/L Summary (0.50 µkat/L). The enzyme aspartate aminotransferase (AST) is widely distributed Icterus:8 No significant interference up to an I index of 60 for conjugated in tissue, principally hepatic, cardiac, muscle, and kidney. Elevated bilirubin and 60 for unconjugated bilirubin (approximate conjugated bilirubin serum levels are found in diseases involving these tissues. Hepatobiliary concentration: 60 mg/dL or 1026 µmol/L; approximate unconjugated bilirubin diseases, such as cirrhosis, metastatic carcinoma, and viral hepatitis concentration: 60 mg/dL or 1026 µmol/L). also increase serum AST levels. Following myocardial infarction, serum Hemolysis:8 No significant interference up to an H index of 40 (approximate AST is elevated and reaches a peak 2 days after onset. hemoglobin concentration: 25.6 µmol/L (40 mg/dL)). In patients undergoing renal dialysis or those with vitamin B6 deficiency, Contamination with erythrocytes will elevate results, because the analyte level serum AST may be decreased. The apparent reduction in AST may be in erythrocytes is higher than in normal sera. The level of interference may be related to decreased pyridoxal phosphate, the prosthetic group for AST, variable depending on the content of analyte in the lysed erythrocytes. resulting in an increase in the ratio of apoenzyme to holoenzyme. 2 isoenzymes of AST have been detected, cytoplasmic and mitochondrial. Lipemia (Intralipid):8 No significant interference up to an L index of 150. There Only the cytoplasmic isoenzyme occurs in normal serum, while the is poor correlation between the L index (corresponds to turbidity) and mitochondrial, together with the cytoplasmic isoenzyme, has been detected triglycerides concentration. in the serum of patients with coronary and hepatobiliary disease. Lipemic specimens may cause > Abs flagging. Choose diluted sample treatment for automatic rerun. Specimen collection and preparation Drugs: No interference was found at therapeutic concentrations For specimen collection and preparation, only use suitable tubes or using common drug panels.9,10 collection containers. Exceptions: Isoniazid can cause artificially low and Furosemide artificially high Only the specimens listed below were tested and found acceptable. AST results at therapeutic concentrations. Serum. Cyanokit (Hydroxocobalamin) may cause interference with results. Plasma: Li-heparin and K2-EDTA plasma In very rare cases, gammopathy, in particular type IgM (Waldenström’s macroglobulinemia), may cause unreliable results. The sample types listed were tested with a selection of sample collection For diagnostic purposes, the results should always be assessed in conjunction tubes that were commercially available at the time of testing, i.e. not all with the patient’s medical history, clinical examination and other findings. available tubes of all manufacturers were tested. Sample collection systems from various manufacturers may contain differing materials which Limits and ranges could affect the test results in some cases. When processing samples in Measuring range primary tubes (sample collection systems), follow the instructions of the 5-700 U/L (0.08-11.7 µkat/L) tube manufacturer. Centrifuge samples containing precipitates before performing the assay. Determine samples having higher activities via the rerun function. Dilution of samples via the rerun function is a 1:10 dilution. Results from samples diluted Stability: 24 hours at 15-25 °C5 using the rerun functionare automatically multiplied by a factor of 10. 7 days at 2-8 °C6 Lower limits of measurement Lower detection limit of the test Traceability 5 U/L (0.08 µkat/L) This method has been standardized against the original IFCC formulation The lower detection limit represents the lowest measurable analyte level using calibrated pipettes together with a manual photometer providing that can be distinguished from 0. It is calculated as the value lying 3 absolute values and the substrate-specific absorptivity. standard deviations above that of the lowest standard (standard 1 + 3 SD, repeatability, n = 21). Quality control Expected values Acc. to the optimized standard method (comparable to the IFCC For quality control, use control materials as listed in the “Order information” method without pyridoxal phosphate activation12): section. In addition, other suitable control material can be used. Males: up to 40 U/L (up to 0.67 µkat/L) Females: up to 32 U/L (up to 0.53 µkat/L) The control intervals and limits should be adapted to each laboratory’s individual requirements. Values obtained should fall within the defined Calculated values: A factor of 2.13 is used for the conversion limits. Each laboratory should establish corrective measures to be taken if from 25 °C to 37 °C.13 values fall outside the defined limits. Each laboratory should investigate the transferability of the expected values Follow the applicable government regulations and local guidelines for to its own patient population and if necessary determine its own reference quality control. ranges. HOOD05162002658134 Effective Date: 9/8/2017 ASTL Aspartate Aminotransferase acc. to IFCC without pyridoxal phosphate activation Precision Precision was determined using human samples and controls in an internal protocol with repeatability* (n = 21) and intermediate precision** (3 aliquots per run, 1 run per day, 20 days). The following results were obtained: Repeatability* Mean SD U/L(µkat/L) C V U/L(µkat/L) % Precinorm U 36.6 (0.611) 0.3 (0.005) 0.8 Precipath U 128 (2.14) 1 (0.02) 0.4 Human serum 1 126 (2.10) 1 (0.02) 0.4 Human serum 2 12.0 (0.200) 0.4 (0.007) 3.1 Intermediate precision** Mean SD U/L(µkat/L) C V U/L(µkat/L) % Precinorm U 36.7 (0.613) 0.5 (0.008) 1.3 Precipath U 130 (2.17) 1 (0.02) 0.8 Human serum 3 30.0 (0.501) 0.7 (0.012) 2.3 Human serum 4 121 (2.02) 2 (0.03) 1.9 * repeatability = within-run precision ** intermediate precision = total precision / between run precision / between day precision Method comparison AST values for human serumand plasma samples obtained on Analyzer Y were compared with those determined using the corresponding reagent on Analyzer X. Sample size (n) = 192 Passing/Bablok Linear regression y = 1.000x - 0.149 U/L y = 0.991x + 1.22 U/L τ = 0.970 r = 0.999 The sample activities were between 30.4 and 674 U/L (0.508 and 11.3 µkat/L). HOOD05162002658134 Effective Date: 9/8/2017