Standard Operating Procedure Subject Bicarbonate (CO2) C311 Index Number Lab-8807 Section Laboratory Subsection Regional/Affiliates Category Departmental Contact Betty Tilleraas Last Revised 10/16/2019 References Required document for Laboratory Accreditation by the College of American Pathologists (CAP), Centers for Medicare and Medicaid Services (CMS) and/or COLA. Applicable To Employees of Gundersen Boscobel Area Hospital laboratory and Palmer Lutheran Hospitals and Clinics laboratories. Detail PRINCIPLE: Bicarbonate reacts with phosphoenolpyruvate (PEP) in the presence of PEPC to produce oxaloacetate and phosphate: PEPC PEP + HCO3- ------- oxaloacetate + H2PO4- The above reaction is coupled with one involving the transfer of a hydrogen ion from NADH analog to oxaloacetate using MDH. MDH Oxaloacetate + NADH analog + H+ --------- malate + NAD+ analog The resultant consumption of NADH analog causes a decrease in absorbance, which is proportional to the concentration of bicarbonate in the sample being assayed. CLINICAL SIGNIFICANCE: The total carbon dioxide content of blood, plasma, or serum consists of an ionized (bicarbonate and carbonate) and a non-ionized (carbonic acid) fraction. The ionized fraction includes HCO3 - , CO23 - , and carbamino compounds. The non-ionized fraction contains H2CO3 and physically dissolved (anhydrous) carbon dioxide. Serum carbon dioxide measurements typically include both of these fractions. Total carbon dioxide content reflects the adequacy of gas exchange in the lungs and the status of acid- base balance. Determination of carbon dioxide concentration in blood is most commonly performed as an initial test in the evaluation of the body’s ability to control blood pH by appropriate removal of metabolism by-products via the lung and kidneys. SPECIMEN: Serum or Li-heparin plasma. (The only acceptable anticoagulant is heparin.) Lab-8807 |Bicarbonate (CO2) C311 Page 1 of 4 Standard Operating Procedure The specimen of choice is plasma from venous blood, collected anaerobically. Serum samples collected anaerobically are also suitable. Tubes must be at least half full. NOTE: The carbon dioxide content of collected human blood is super-saturated relative to the atmosphere and is therefore susceptible to rapid loss. To minimize carbon dioxide loss, specimens should be kept tightly capped and refrigerated until just before analysis. The loss of carbon dioxide concentration of uncapped serum or plasma tubes is approximately 4 mmol/L after 1 hour. Specimen Storage and Stability: Seven days at 2-8 °C when separated from erythrocytes and stored tightly stoppered. NOTE: Test requests for CO2 may only be added to specimens that have not been opened/uncapped. REAGENTS/MATERIALS: R1 Phosphoenolpyruvate: ≥ 40 mmol/L; NADH analog: ≥ 2 mmol/L; MDH (porcine): ≥ 314.3 µkat/L; PEPC (microbial): ≥ 30.8 µkat/L R1 is in position B. Reagent Precautions: Exercise the normal precautions required for handling all laboratory reagents. Reagent Storage and Stability: CO2-L Shelf life at 2-8 °C: See expiration date on cobas c pack label. On-board in use and refrigerated on the analyzer: 6 weeks EQUIPMENT/INSTRUMENTATION: Cobas C311 Calibration: Calibrators S1: H2O S2: Ammonia/Ethanol /CO2 Calibrator Calibration model Linear Calibration Frequency: 2-point calibration The instrument should be calibrated: 1. at initial installation 2. if necessary after instrument service or repair 3. if dictated by quality control results 4. if instructed to do so by Roche support personnel 5. with each new reagent lot # Calibrator Preparation, Storage, and Procedure Lab-8807 |Bicarbonate (CO2) C311 Page 2 of 4 Standard Operating Procedure Refer to the COBAS c311 User Manual located in the laboratory. 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 Implementation PROCEDURE NOTES: AMR (Analytic Measurement Range): 2.0 – 50 mmol/L No post dilution Report values greater than 50 as > 50mmol/L. Report values less than 2 as < 2mmol/L. CALCULATIONS: N/A INTERPRETATION: Results Reporting: C02 is reported in whole numbers in mmol/L. Expected values: 22-29 mmol/L LIMITATIONS: Criterion: Recovery within ± 10 % of initial value at a bicarbonate concentration of 22 mmol/L. Icterus: No significant interference up to an I index of 60 for conjugated and unconjugated bilirubin (approximate conjugated and unconjugated bilirubin concentration: 1026 µmol/L or 60 mg/dL). Hemolysis: No significant interference up to an H index of 600 (approximate hemoglobin concentration: 372.6 µmol/L or 600 mg/dL). Lipemia (Intralipid): No significant interference up to an L index of 1800. There is poor correlation between the L index (corresponds to turbidity) and triglycerides concentration. Drugs: No interference was found at therapeutic concentrations using common drug panels. In very rare cases, gammopathy, in particular type IgM (Waldenström'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. Lab-8807 |Bicarbonate (CO2) C311 Page 3 of 4 Standard Operating Procedure An abnormally elevated concentration of ambient carbon dioxide (CO2) may occur under certain environmental conditions in the laboratory. The fluctuating ambient CO2 concentration may interfere with the CO2-L assay leading to higher CO2 results. Under these circumstances, the reduction of the re- calibration interval may become necessary if the laboratory is unable to keep the ambient CO2 concentration at a normal level by appropriate countermeasures. Special Wash Programming: The use of special wash steps is mandatory when certain test combinations are run together on Roche/Hitachi cobas c systems. The latest version of the carry-over evasion list can be found with the NaOHD/SMS/Multiclean/SCCS or the NaOHD/SMS/SmpCln1 + 2/SCCS Method Sheets. For further instructions refer to the operator's manual. Where required, special wash/carry-over evasion programming must be implemented prior to reporting results with this test. REVIEW AND CHANGES: This document and all attached forms should be reviewed optimally on an annual basis, with two years as the maximum review date. Review will be done by the Technical Leader, Supervisor, Manager, Medical Director or other designated person. Changes require retyping the document, and review by the Medical Director. REFERENCES: Roche COBAS C311 Implementation User’s Manual Roche COBAS C311 Carbon Dioxide Method Sheet. Roche COBAS C311 Method Manual. Lab-8807 |Bicarbonate (CO2) C311 Page 4 of 4 .