Standard Operating Procedure

Transcription

1 Subject Cholestech Index Number Lab-8788 Section Laboratory Subsection Regional Clinic/Affiliate Hospital Laboratories Category Departmental Contact Karen Munson Last Revised 12/12/2017 References Required document for Laboratory Accreditation by the College of American Pathologists (CAP), Centers for Medicare and Medicaid Services and/or COLA. Applicable To Employees of Gundersen St. Joseph s Hospital Laboratories Detail PRINCIPLE: The Cholestech LDX System combines enzymatic methodology, solid-phase technology reflectance photometry to measure total cholesterol, HDL cholesterol, triglycerides, and glucose. The Cholestech cassette separates the plasma from the blood cells. A portion of the plasma flows to the right side of the cassette and is transferred to both the total cholesterol and triglyceride reaction pads. Simultaneously, plasma flows to the left side of the cassette where the low-density lipoproteins (LDL and VLDA) are precipitated with dextran sulfate ( MW) and magnesium acetate precipitating reagent. The filtrate, containing both the glucose and HDL cholesterol, is transferred to both the glucose and HDL cholesterol reaction pads. The Cholestech LDK Analyzer measures total cholesterol and HDL cholesterol by an enzymatic method based on the formulation of Allain et al, and Roeschlau. Cholesterol esterase hydrolyzes the cholesterol ester in the filtrate or plasma to free cholesterol and the corresponding fatty acid. Cholestero oxidase, in the presence of oxygen, oxidizes free cholesterol to cholest- 4-ene-3-one and hydrogen peroxide. In a reaction catalyzed by horseradish peroxidase, the peroxide reacts with 4-Aminotipyrine and N-Ethyl-Nsulfohydorxypropyl-m-toluidine, sodium salt (TOOS) to form a purple-colored quinoneimine dye proportional to the total cholesterol and HDL cholesterol concentrations of the sample. Cholesterol Esters + H2O Cholesterol esterase > Free cholesterol + Fatty Acids Cholesterol + O2 Cholesterol oxidase > Cholest-4-ene-3-one + H2O2 2H2O2 +4-Aminoantipyrine + TOOS Peroxidase > Quinoneimine dye + H2O The Cholestech LDX Analyzer measures triglycerides by an enzymatic method based on the hydrolysis of triglycerides by lipase to glycerol and free fatty acids. Glycerol, in a reaction catalyzed by glycerol kinase, is converted to glycerol-3-phosphate. In a third reaction, glycerol-3-phosphate is oxidized by glycerol phosphate oxidase to dihydroxyacetone phosphate and hydrogen peroxide. The color reaction utilizing horseradish peroxidase is the same as the total cholesterol and HDL cholesterol. Triglycerides + H2O Lipase > Glycerol + Free fatty acids Lab-8788 Cholestech Page 1 of 9

2 Glycerol + ATP Glycerol kinase + Mg2+ > Glycerol-3-hosphate + ADP Glycerol-3-phosphate + O2 Glycerol phosphate > Dihydroxyacetone phosphate + H2O2 2 H2O Aminoantipyrine + TOOS Oxidase Perosidase> Quinoneimine dye + 4 H2O The Cholestech LDX Analyzer measures glucose by an enzymatic method that uses glucose oxidase to catalyze the oxidation of glucose to gluconolactone and hydrogen peroxide. The color reaction utilizing horseradish peroxidase is the same as that for total cholesterol, HDL cholesterol and triglycerides. Glucose + O2 Glucose oxidase> o-d-gluconolactone + H2O2 2 H2O2 + 4-Aminoantipyrine + TOOS Peroxidase> Quinoneimine dye + 4 H2O The resultant color of all four reactions is measured by reflectance photometry. The Cholestech LDX analyzer converts the reflectance reading (%R) of each test to concentrations in mg/dl. CLINICAL SIGNIFICANCE: Cholestech Measurements of serum cholesterol levels are important in the diagnosis and classification of hyperlipoproteinemias. Elevated cholesterol levels may occur with hypothyroidism, nephritic syndrome, diabetes, and various liver diseases. There is correlation between elevated serum cholesterol levels and the incidence of coronary artery diseases. Normal cholesterol levels are affected by stress, diet, age, gender, hormonal balance and pregnancy. Depressed levels are associated with hyperthyroidism and severe liver disease. HDL Cholesterol High density lipoproteins (HDL) are responsible for reverse transport of cholesterol from the peripheral cells to the liver. Here, cholesterol is transformed to bile acids, which are excreted into the intestine via the biliary tract. Monitoring of HDL cholesterol in serum is of clinical importance since the inverse correlation exists between HDL cholesterol concentrations and the risk of atherosclerotic disease. Elevated HDL cholesterol concentrations are protective against coronary heart disease, while reduced HDL cholesterol concentrations, particularly in conjunction with elevated triglycerides, increase the risk of cardiovascular risk. Triglycerides Triglycerides are the main lipids present in human plasma; the others are cholesterol, phospholipids, and non-esterfied fatty acids. They are transformed in the intestinal mucosa by the esterification of glycerol and free fatty acids. They are then released into the mesenteric lymphatics and distributed to most tissues for storage lipids in humans, where thye constitute about 95% of adipose tissue lipids. Elevated levels of triglycerides have been associated with high risk in severe atherosclerosis. High triglycerides levels and hyperlipidemia in general can be an inherited trait or can be secondary to disorders including diabetes mellitus, nephrosis, biliary obstruction and metabolic disorders associated with endocrine disturbances. Glucose Glucose is the major carbohydrate present in the peripheral blood. Oxidation of glucose is the major source of cellular energy in the body. Glucose derived from dietary sources is converted to glycogen for storage in the liver or to fatty acids for storage in adilpose tissue. The concentration of glucose in blood is controlled within narrow limits by many hormones, the most important of which are produced by the pancreas. The most frequent cause of hyperglycemia is diabetes mellitus resulting from a deficiency in insulin secretion or action. A number of secondary factors also contribute to elevated blood glucose levels. These include pancreatitis, thyroid dysfunction, renal failure, and liver disease. Lab-8788 Cholestech Page 2 of 9

3 Hypoglycemia is less frequently observed. A variety of conditions may cause low blood glucose such as insulinoma, hypopituitarism, or insulin-induced hypoglycemia. Objectives: 1. To provide guidelines for the use of Choestech LDX System in a waived laboratory or non-laboratory setting. 2. To establish uniform testing and reporting guidelines. 3. To assure that only certified operators perform this test. All certification/credentialing is conducted in accordance with Lab-0955 Point of Care Testing General Policy/Quality Assurance described specifically under the Testing Personnel section. SPECIMEN: 40 microliters of capillary whole blood collected into a Choestech Capillary Tube (lithium heparin anticoagulant). Specimen Identification: Patient identification occurs by either verbal verification of the patient s name and birthdate (outpatient setting), or the verification of the patient s name and clinic number found on the patient s ID armband (inpatient setting). Refer to Lab-1330 Patient Identification for procedural guidelines. Specimen Collection: All samples are collected by skin puncture following the standard precautions outlined in Lab-0105 Safety: Biological and the steps outlined below. 1. Insert a capillary plunger into a Cholestech capillary tube at the end with the red mark. Set it aside. 2. Choose a spot on the side of one of the center fingers of either hand. The fingers and hands should be warm to the touch. To warm hand, you can: Wash the patient s hand with warm water or apply a warm compress to the hand for several minutes, or gently massage the finger from the base to the tip several times to bring the blood to the fingertip. 3. Clean the site with an alcohol wipe and allow to dry thoroughly. 4. Firmly prick the selected site with the lancet device. 5. Wipe away the first drop of blood as it may contain tissue fluid. 6. Squeeze the finger gently again to form a second large drop of blood. Do not milk the finger. Use dependent hand and arm portion to promote blood flow to the fingertip. The puncture should provide a free-flowing drop of blood. 7. Hold the capillary tube horizontally by the end with the plunger. Touch it to the drop without touching the skin. The tube will fill by capillary action up to the black mark. Do not collect air bubbles. Continue repeating step 7 until the capillary tube is filled to the black mark. Stability: Blood collected in the capillary tube must be placed into the test cassette within five minutes of collection. Unacceptable Specimens: Lab-8788 Cholestech Page 3 of 9

4 1. Clotted specimens and hematocrits less than 20% and higher than 80% may affect migration of the blood through the cassette and yield Reaction Did Not Occur error messages. 2. Hand creams and soaps with glycerol may cause falsely high triglyceride results. 3. Alcohol contaminated specimens yield inaccurate results. 4. Reactions will not occur with a QNS sample. REAGENTS/MATERIALS: Cholesterol LDX Test Cassettes, Alere: The cassette can be purchased in a variety of test combinations. Store the cassette in the manufacturer sealed pouches at 2-8 C. Do not allow the cassettes to freeze. Refrigerated cassettes are stable until the expiration date printed on each sealed pouch. Cassettes may be stored at room temperature (less than 86 F/30 C) with a modified 30-day expiration date. Do not use any cassette beyond the expiration date (manufacturer or modified). Cassettes should not be returned to the refrigerator once equilibrated to room temperature. Do not reuse assayed cassettes. Cholestech Multianalyte Level 1 and Level 2 Lipid Controls, Alere: Store control vials upright and refrigerated at 2-8 C. Unopened, refrigerated controls are stable until the expiration date printed on the box. Opened control vials are stable for 30 days refrigerated at 2-8 C. Minimize exposure to light. Do Not Freeze. Allow the control vials to warm at room temperature for 10 minutes prior to use. Cholestech Calibration Verification Material, Levels 1-4, Alere: Store vials refrigerated at 2-8 C. Open vial stability is 30 days at 2-8 C. Do Not Freeze. Minimize exposure to strong light. Cholestech LDX Optics Check Cassette, Alere: Store the Cholestech LDX Optics Check Cassette at room temperature in the case provided. Do not touch the reaction bar or allow it to become wet, dirty or scratched. Do not use a damaged or expired Cholestech LDX Optics Check Cassette. Other material needed: Cholestech LDX capillary tubes and plungers (with lithium heparin anticoagulant) LDX Axihom Label Rolls (if attached to the external printer) Alcohol Wipes 2 x 2 sterile gauze pads Disposable vinyl gloves Disposable Lancet device 40 microliter MiniPet (fixed volume) and pipette tips EQUIPMENT/INSTRUMENTATION: Cholestech LDX Test System, Alere: Includes Cholestech Analyzer, Printer (or Computer), and Power Source. Calibration: Instrument calibration/verification is performed by the manufacturer and validated initially on each new/repaired analyzer by using Cholestech Calibration Verification Material Level 1-4; and supplemental Lab-8788 Cholestech Page 4 of 9

5 patient plasma samples as needed. The magnetic brown stripe on each test cassette contains specific lot calibration data and is utilized each time a cassette is run. The analytical measurement range is validated using a low, mid and high analyte tested in duplicate and compared to the acceptable ranges established by the manufacturer. QUALITY CONTROL: The Cholestech quality control program is designed to verify the performance of the analyzer and test cassettes. Quality control frequency is defined as once on each date of use and always prior to patient testing. Optics Check Cassette Quality Control: The Optics Check Cassette with known reflectance values is used to check the optics of each analyzer once per day before samples are tested and after the Cholestech LDX System has been moved or serviced. The assigned values are stamped on each Optics cassette. Record the results in the Optics Check Daily Log. 1. Press the RUN button. After verifying the Selftest OK message, the drawer will open and the screen will display Load Cassette and press RUN. 2. Place the Optics Check Cassette into the cassette drawer. NOTE: Do not place blood sample into this cassette. 3. Press RUN button again and the analyzer will automatically perform the optics check. The word Optics Check and four numbers will appear on the screen, one for each optical channel in the analyzer. 4. If the results of all four channels fall within the ranges printed on the Optics Check Cassette label, the system is ready for use. 5. If the values for any of the channels fall outside the ranges printed on the Optics Check Cassette label, the analyzer will shut down. The analyzer will be disabled until another optics check has been run that falls within range. Repeat the test with a different Optics Check Cassette. If it sill fails, call Alere Technical Service at , Option Record the results in the Optics Check Daily Log. Liquid Quality Control: Preparation and Handling: Cholestech LDX Multianalyte Liquid Control solutions, Level 1 and Level 2, come ready to use in small screw-capped vials. Refrigerated control vials must equilibrate to room temperature for about 10 minutes prior to use. Verify that the lot number on the control vial and the assay sheet are the same. Mix each vial 7-8 times immediately before use. Store the controls in the refrigerator when not in use. The liquid quality controls are used to validate each new lot # shipment of cassettes prior to patient use, in accordance with manufacturer guidelines. Stepwise Instructions: 1. Remove a test cassette from its foil pouch and place on a flat, dry surface. 2. Press the RUN button on the analyzer. The cassette drawer will open, and the screen will display, Load Cassette and Press Run. 3. Use the MiniPet Pipette and tips to measure and dispense the control sample into the cassette s center sample well. Lab-8788 Cholestech Page 5 of 9

6 4. Keep the cassette flat after the sample has been applied. Place the cassette in the drawer immediately. (Note: The black reaction bar must face the back of the analyzer. The brown stripe must be on the right). 5. Press RUN. The drawer will close. During the test, the screen will display Test Name (s) and Test Running. 6. When the test is complete, the analyzer will beep. The screen will display the test name, results and warnings (if error occurred in the test). Press DATA to show more results. 7. When the drawer opens, discard the cassette into the appropriate biohazard container. 8. Record results on the quality control log sheet. The results must fall within the expected range for each analyte before patient testing can occur. 9. Repeat steps 3-9 for the next level or to repeat the same level. Quality control results are documented on the Quality Control Log sheets provided for each level. All control results must fall within the stated range before patient testing may take place. If test results fall outside the acceptable range, corrective action steps must be initiated until resolution is achieved. Document all corrective action steps taken on the Quality Control Log sheet. Common Errors Causing QC to Fail: 1. Failure to allow cassette or control material to equilibrate to room temperature for ten minutes. 2. Failure to mix the control adequately prior to testing. 3. Delays in placing the cassette into the cassette drawer immediately. Liquid Quality Control data is reviewed prior to patient use by the POCC or designee. Optic check Cartridge QC is reviewed once per month by the POCC or designee. It is reviewed and corrective action initiated in accordance to Lab-0170 Quality Control and Lab-0135 Quality Assurance for Laboratory Testing. Quality Assurance: Lab POC staff will validate that each lot of liquid QC test values fall within the manufacturer stated range. Prior to use, each new lot number is tested with both levels of liquid quality control in order to validate performance. Cassettes will not be released for testing if performance levels are not achieved and the manufacturer will be contacted for replacement. Proficiency Testing (Non-Waived Labs only): Point of Care Testing Liquid Profile and Glucose Option using Cholestech LDX System is enrolled in the CAP Proficiency Testing Program. The lab director or designee reviews the results. Lab-0135 Quality Assurance for Laboratory Testing is initiated for corrective action if results exceed tolerance limits. Implementation PROCEDURE: 1. Let the test cassette warm at room temperature for a minimum of 10 minutes. 2. Remove the cassette from its foil pouch. Do not touch the black reaction bar or the brown information stripe. Place the cassette on a flat surface. 3. Press RUN. In a few seconds the screen will read Selftest running. Selftest OK. 4. The cassette drawer will open. The screen will read Load cassette and press RUN. Lab-8788 Cholestech Page 6 of 9

7 5. Collect a capillary specimen following the steps outlined in the Specimen Collection section of this policy. 6. Place the capillary tube tip into the cassette well. NOTE: Fingerstick samples must be applied to the cassette within five minutes. 7. Keep the cassette flat after the sample has been applied. Place the cassette into the drawer of the Cholestech analyzer at once. The black bar must face the back of the analyzer and the brow strip must be on the right. 8. Press RUN. The drawer will close. During the test the screen will read: Test Name (s) Test Running***. The stars displayed on the screen indicate elapsed time toward test completion. 9. Discard capillary collection material into the appropriate biohazard collection containers. 10. When the test is complete, the Cholestech analyzer will make an audible beep. The screen will read: Test Name (s) = ### (results). 11. Press DATA to display more test names and results. NOTE: When test results fall outside the measuring range, greater than > or less than < symbol displays by the test name and test result. 12. Error messages will also display on the screen. Please refer to the Cholestech LDX System manual for additional information or corrective action suggestions. 13. Record the results per department protocol. 14. Discard used cassette into the appropriate biohazard container. 15. To run another cassette, press RUN. The screen will once again read Load cassette and press RUN. Remove the cassette from its foil pouch. Do not touch the black reaction bar or the brown information stripe. Place the cassette on a flat surface. 16. Repeat the test procedure, steps 5-14, for the next patient sample. PROCEDURE NOTES: 1. Hand creams and soaps with glycerol may cause falsely elevated triglyceride results. Cleanse the test site completely before collection to eliminate this interfering substance. 2. Squeeze the capillary puncture site excessively may cause erroneous test results due to hemolysis or dilution of the specimen with tissue fluids. 3. Samples that yield results outside the measuring range are sent to a laboratory for confirmatory testing or the patient is consulted by screening personnel for further options. 4. The Cholestech has two testing modes, Whole Blood or Serum. Capillary whole blood samples and quality control materials are tested in the whole blood mode. Consult the System User Manual for the complete process to change testing modes. 5. Once the Load cassette and Press RUN screen is displayed, the analyzer will wait in the ready status for four minutes. At four minutes a beep will sound and the screen will display: System timeout RUN to continue. If the RUN button is not pushed within 15 seconds, the drawer will close and the screen will go blank. 6. If necessary press the DATA button to view the results from the last test cassette tested. 7. To avoid drawer jams, the cassette must be placed flat in the cassette drawer. 8. The Cholestech LDX analyzer contains a battery that cannot be replaced by the user. To conserve this battery, plug the analyzer into a power source when not in use. 9. NOTE: Do not remove the ROM pack while the Cholestech analyzer is plugged into a power source. The analyzer will lose its optical calibration and will no longer function. CALCULATIONS: Lab-8788 Cholestech Page 7 of 9

8 The LDL Cholestech is calculated from the total cholesterol, HDL cholesterol and triglyceride tests result using the following equation: Friedwald Formula LDL Cholesterol = Total Cholesterol [(Trig x 0.20) + HDL Cholesterol] INTERPRETATION: Cholesterol Expected Range: DESIRABLE Adult <200 mg/dl <18 years <170 mg/dl Analytical Measurement Range: mg/dl. Results outside this range will appear as <100 mg/dl or >500 mg/dl. HDL Cholesterol: Expected Range: mg/dl Analytical Measurement Range: mg/dl. Results outside this range will appear as <15 mg/dl and >100 mg/dl. Glucose: Expected Range (Fasting): mg/dl Analytical Measurement Range: mg/dl. Results outside this range will appear as <50 mg/dl and >500 mg/dl. Triglyceride Expected Range: Age Male Female mg/dl mg/dl mg/dl mg/dl mg/dl mg/dl > mg/dl mg/dl Analytical Measurement Range: mg/dl. Results outside this range will appear as <45 mg/dl or >650 mg/dl. Reporting: As used in the health screen environment, the Cholestech LDX Analyzer can utilized a variety of reporting options: patient result label, cabling to a computer PC loaded with a Coronary Risk Assessment Program, or manual recording onto a result log sheet. Results are distributed to the patient in the manner designed by testing site or situation, varying from screen to screen. The POC lab will maintain a copy of this record for troubleshooting and detection of clerical error. LIMITATIONS: 1. If the triglycerides are >400 mg/dl, the estimated LDL will not be calculated. 2. If the total cholesterol, HDL cholesterol or triglyceride result is outside the preset measuring range, the LDL will appear as N/A. Lab-8788 Cholestech Page 8 of 9

9 3. If triglycerides are >650 mg/dl, the HDL cholesterol result may not be accurate and will appear as N/A. 4. Hematocrits less than 30% and greater than 52% potentially affect the migration of the blood through the cassette and yield a Reaction Did Not Occur error message. These patients should be tested by laboratory methods. 5. The glucose test is specific for D-glucose. Other sugars that may be present in the blood do not react in the glucose test (i.e. fructose, lactose). 6. Patient samples that yield results outside the measuring range are sent to a laboratory for confirmatory testing or the patient is consulted by screening personnel for further options. 7. Refer to the product insert for a complete list of interfering substance that may cause false results with enzymatic tests. The substances on this list demonstrated less than 10% interference at the levels shown. 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. Cholestech LDX System User Manual, Cholestech Corporation, 2004, Revision C. 2. Cholestech LDX System Procedure Manual, CD-ROM, Adobe Acrobat, PDF Version. 3. Cholestech LDX Test Cassette Product Insert, Gundersen Lutheran Lab-4285 Cholesterol, Lab-4335 Glucose, Lab-4287 HDL Cholesterol, and Lab-4455 Triglycerides. Lab-8788 Cholestech Page 9 of 9