Standard Operating Procedure

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1 Subject Alkaline Phosphatase C111 Index Number Lab-1504 Section Laboratory Subsection Regional Clinic/Affiliate Hospital Laboratories Category Departmental Contact Kamprud, Elizabeth Last Revised 4/12/2017 References Required document for Laboratory Accreditation by the College of American Pathologists (CAP), Centers for Medicare and Medicaid Services (CMS) and/or COLA. Employees of GHS clinical laboratories. Applicable To Detail The cassette Cobas c 111 Alkaline phosphatase/alp (ALP2S) contains an in vitro diagnostic reagent system intended for use on Cobas systems for the quantitative determination of the ALP (ALP2S) concentration in serum and plasma. PRINCIPLE: Colorimetric assay in accordance with a standardized method. In the presence of magnesium and zinc ions, p-nitrophenyl phosphate is cleaved by phosphatases into phosphate and p-nitrophenol. The p-nitrophenol released is directly proportional to the catalytic ALP activity. It is determined by measuring the increase in absorbance. CLINICAL SIGNIFICANCE: Alkaline phosphatase refers to a group of phosphates (ph optimum approximately 10) found in almost every tissue in the body. Most alkaline phosphatase in normal adult serum is from the liver or biliary tract. Normal alkaline phosphatase levels are age dependent with young children and adolescents having much higher levels than adults. Adult males tend to have higher levels than females, but pregnant females have increased levels due to placental secretion of alkaline phosphatase. Elevation of alkaline phosphatase levels occurs in diseases such as hepatitis, cirrhosis, malignancy, chemical toxicity, and in bone diseases such as metastatic carcinoma, rickets, Paget s disease, and osteomalacia. Moderate increases in serum alkaline phosphate levels have been observed in Hodgkin s disease, congestive heart failure, ulcerative colitis, regional enteritis, and intra-abdominal bacterial infections. Normal alkaline phosphatase levels are elevated during periods of active bone growth, for example, in young children and adolescents. SPECIMEN: Universal Precautions apply Page 1 of 5

2 Serum or Plasma The only acceptable anticoagulant is Li-heparin. Stability in serum/plasma: 7 days at o C, 7 days at 2-8 o C, 2 months at o C REAGENTS/MATERIALS: Cobas C111- Alkaline phosphatase 50 tests Cassette ALP2S R1 2-Amino-2-methyl-1-propanol: mol/l, ph (30 o C); magnesium acetate: 3.83 mmol/l; zinc sulfate: mmol/l; N-(2-hydroxyethyl)-ethylenediamine triacetic acid: 3.83 mmol/l SR p-nitrophenyl phosphate: mmol/l, ph 8.5 (30 o C); preservatives Reagent Handling: Ready to use. Place a chimney in R1 before use. Absorption of atmospheric CO 2 by the opened reagent bottle R1 leads to impaired reagent stability. This kit therefore requires the use of chimneys which reduce the uptake of CO 2 by the reagent. The chimneys should be placed directly into the appropriate reagent. The chimneys can be reused for reagent bottles within the same kit. Do not wash the chimneys before re-use. Storage and Stability: Shelf life at 2-8 o C: see expiration date on cassette. On-Board in use at 2-8 o C: 10 days. EQUIPMENT/INSTRUMENTATION: Cobas c111 Calibration: Calibrators: Calibrator f.a.s. Deionized water is used automatically by the instrument as the zero calibrator. Calibration mode: Linear regression Calibration interval: 1. Each lot 2. Every 5 days 3. As required following quality control procedures Traceability: This method has been standardized against the original IFCC formulation using calibrated pipettes together with a manual photometer providing absolute values and the substrate-specific absorptivity, Ƹ. 6 QUALITY CONTROL: Quality Control Frequency: Two levels of quality control should be performed at a minimum: 1. Once every twenty-four hours 2. If a new cassette of reagent is put in use 3. If a calibration is performed Refer to Lab-0170 Quality Control. Page 2 of 5

3 Implementation Refer to the Cobas c 111 User Manual located in the chemistry department. PROCEDURE NOTES: AMR: (Analytical Measurement Range): U/L Report values of greater than AMR as >1200 U/L CALCULATIONS: The cobas c 111 analyzer automatically calculates the analyte activity of each sample. Conversion factor: U/L x = ukat/l INTERPRETATION: Expected Values Adults: Males U/L Females U/L Children: Males Females 0-14 days days - < 1 year < 10 years < 13 years < 15 years < 17 years < 19 years LIMITATIONS: Criterion: Recovery within + 10% of initial value at an alkaline phosphatase activity of 129 U/L (2.15 ukat/l). Icterus: No significant interference up to an I index of 60 for conjugated and unconjugated bilirubin (approximate conjugated and unconjugated bilirubin concentration: 1026 umol/l (60 mg/dl)). Hemolysis: No significant interference up to an H index of 250 (approximate hemoglobin concentration: 155 umol/l (250 mg/dl)). Lipemia (Intralipid): No significant interference up to an L index of There is poor correlation between the L index (corresponds to turbidity) and triglycerides concentration. Drugs: No interference was found using common drug panels. 9 Page 3 of 5

4 In very rare cases gammopathy, in particular type IgM (Waldenstrom s macroglobulinemia), may cause unreliable results. For diagnostic purposes, the results should always be assessed in conjunction with the patient s medical history, clinical examination and other findings. Special wash requirements No interfering assays are known which require special wash steps. Method comparison: Alkaline phosphatase values for human serum and plasma samples obtained on a cobas c 111 analyzer (y) were compared with those determined using the corresponding reagent on a COBAS INTEGRA 400 analyzer (x). Sample size (n) = 74 Passing/Bablok 13 Linear regression y = 0.997x U/L y= 1.008x U/L t= r= The sample activities were between 32 and 828 U/L (0.534 and 13.8 ukat/l). REVIEW AND CHANGES: This document and all attached forms should be reviewed optimally on an annual basis, with 2 years as the maximum review date. Review will be done by the Technical Leader, Supervisor, Manager, Medical Director or designated person. Changes require retyping document or form and review by the Medical Director. REFERENCES: 1. Alkaline Phosphatase acc. to IFCC Gen.2 Method sheet. (ALP25) February Greiling H, Gressner AM, eds. Lehrbuch der Klinischen Chemie und Pathobiochemie, 3 rd ed. Stuttgart/New York: Schattauer Verlag, King EJ, Armstrong AR. Can Med Assoc J 1934; 31: Ohmori Y. Enzymologia 1937; 4: Bessey OAH et al. J Biol Chem 1946; 164: Hausamen TU et al. Clin Chem Acta 1967; 15: Tietz NW et al. J Clin Chem Clin Biochem 1983; 21: Use of Anticoagulants in Diagnostic Laboratory Investigations. WHO Publication WHO/DIL/LAB/99.1 Rev Glick MR, Ryder KW, Jackson SA. Graphical Comparisons of Interferences in Clinical Chemistry Instrumentation. Clin Chem 1986; 32: Report on the Symposium Drug effects in clinical chemistry methods: Breuer J, Eur J Clin Chem Clin Biochem 1996; 34: Abicht K et al. Multicenter evaluation of new GGT and ALP reagents with new reference standardization and determination of 37 o C reference intervals. Clin Chem Lab Med 2001; 39 (supply.): S346 (abstract). 12. Thomas L, Muller M, Schumann G, Weideman G et al. Consensus of DGKL and VDGH for interim reference intervals on enzymes in serum. J Lab Med 2005; 29: Page 4 of 5

5 13. Fischbach F, Zawta B. Age-dependent Reference Limits of Several Enzymes in Plasma at Different Measuring Temperatures. Klin Lab 1992; 38: Bablok W et al. A General Regression Procedure for Method Transformation. J Clin Chem Clin Biochem 1988; 26: Page 5 of 5