Reviewed by. Joseph V. Bonventre. Document History Date Comment Reviewer Signature 12/20/17 Updated SOP Venkata Sabbisetti

Transcription

1 BWH: Center for Kidney Diseases HIM 550, Harvard Institutes of Medicine 77 Louis Pasteur Avenue, Boston, MA Phone # Date: March 5, 2018 Title: human urinary 2 Plex using Randox Daytona Reference: human urine 2 Plex SOP Version Number: 1.1 Effective Date: 4/19/17 Review Date: effective +4 years Author Venkata Sabbisetti Reviewed by Joseph V. Bonventre Title Instructor in Medicine Title Chief, Renal Division Signature Signature Date Date Document History Date Comment Reviewer Signature 12/20/17 Updated SOP Venkata Sabbisetti 03/08/28 Updated SOP Venkata Sabbisetti Author: Venkata S. Sabbisetti Page 1 of 11

2 1. Purpose Introduction... Error! Bookmark not defined. 3. Sample Collection, Preperation & Storage Procedure Location Required equipment / reagents Buffer preparation Microbead preparation Experimental Procedure Equipment Plate layout Data Generation & Processing Data output Standard curve generation Data processing and caliculations Quality Control Quality control of the assay Quality control of each sample Quality control trouble shooting procedure Data Storage & Reporting Plex assay parameters References Product inserts Purpose This Standard Operating Procedure (SOP) describes the process for the quantitative assessment of Urinary Creatinine (UCr) and Urine Phosphorus in human urine specimens. These can be measured simultaneously or individually in urine specimens on Randox Daytona Platform 2. Introduction This SOP describes for assays to measure Phosphorus and Creatinine using Randox Daytona Instrument. Author: Venkata S. Sabbisetti Page 2 of 11

3 3. Sample Collection, Handling & Storage Frozen urine samples received from investigators by the central laboratory will be immediately stored at 80 C. Urine creatinine and phosphorus are stable for up to at least 5 freeze thaw cycles. All the personnel handling the specimens will have undergone Environmental Health & Safety training at Brigham & Women s Hospital. Protective gear including lab coats and gloves must be worn while working in the laboratory. 4. Procedure 4.1. Location Harvard Institutes of Medicine Room 550 Renal Division/ Department of Medicine/ Brigham and Women s Hospital 4.2. Required equipment / reagents Randox Daytona Refrigerated Centrifuge (Eppendorf 5430R Rotor F ) MilliQ water (Type 1, reagent grade water) Refrigerated Centrifuge (Eppendorf 5430R) Vortexer (Denville Scientific) Single Channel Manual Pipettes o Rainin Classic 0.5 μl-10 μl (catalog # PR-10) o Rainin Classic 10 μl-100 μl (catalog # PR-100) o Rainin Classic 100 μl-1000 μl (catalog # PR-1000) 30C incubator (Thermo Scientific) 4.3. Randox Reagent Preparation Assay Reagents Preparation Inorganic Phosphorus (PHOS) Cat # PH3872 Creatinine (CREA) Cat # CR3814 R1a. Blank Reagent R1b. Molybdate R1. Sodium Hydroxide R2. Picric acid Pour the contents of 1 vial of R1b in bottle of R1a. Mix by inversion. Reconstituted reagent is stable for up to 8 weeks. Reagents are ready to use. Once open, the reagents are stable for up to 21days Preparation of other reagents Assay Calibrants Preparation Inorganic Phosphorus (PHOS) Creatinine (CREA) Calibration Serum Level 3 (Cat # CAL2351) Randox Calibration Serum Level 2 (Cat # CAL2351) and Calibration Serum Level 3 (Cat # CAL2351) Add 5 ml of Milli Q water to bottle and mix well by shaking the bottle. Once prepared calibrant is stable for a week at 4C. Add 5 ml of Milli Q water to each bottle and mix well by shaking the bottle. Once prepared calibrants are Author: Venkata S. Sabbisetti Page 3 of 11

4 stable for a week at 4C. Assay Controls Preparation Inorganic Phosphorus (PHOS) Creatinine (CREA) Assayed Urine Control- Level 2 (AU 2352) Assayed Urine Control- Level 3 (AU 2353) Assayed Urine Control- Level 2 (AU 2352) Assayed Urine Control- Level 3 (AU 2353) Add 10 ml of Milli Q water to each bottle and mix well by shaking the bottle. Once prepared Controls are stable for a week at 4C. Add 10 ml of Milli Q water to each bottle and mix well by shaking the bottle. Once prepared Controls are stable for a week at 4C. Other Reagents: Rx Series Saline (Cat. No. SA 3854). This will be used for urine dilution as well as blank in the calibration of the assays. Assay Principles: Urine Phosphorus (UV method): Inorganic phosphorous reacts with ammonium molybdate in the presence of sulphuric acid to form a phosphomolybdate complex which is measured at 340 nm. Urine Creatinine (Colorimetric method): Creatinine in alkaline solution reacts with picrate to form a colored complex. The rate of formation of the complex is measured Experimental Procedure Sample thawing: Sample tubes will be arranged in open tube racks with an empty space between each tube for better air circulation. Place these racks in 30 C incubator until all samples are thawed (The tubes will be still cold after the samples are thawed). Vortex the tubes for 5 sec, invert the tube for 5 times, centrifuge at 1800 x g for 5 min at 4 C, keep on ice, and proceed for the analysis. Quality Controls that contain high and low urine Biomarker values are prepared as described above Prepare Calibrants as described above. Generation of calibration curve for PHOS Assay: This assay uses a linear calibration. Saline solution will be used as zero calibrator and Calibration Serum Level 3 as second calibrator. Generation of calibration curve for Creatinine Assay: This assays uses a linear calibration. Calibration Seral Level 2 and Level 3 is recommended for calibration. 4.6 Equipment Randox Daytona Machine will be calibrated for each assay every week. 4.7 Sample and Reagent layout in the Randox Daytona instrument Author: Venkata S. Sabbisetti Page 4 of 11

5 Initiation of Randox Daytona. Randox Daytona has two carousals: Reagent Container Unit and Auto Sampler Unit. Reagent container is refrigerated and keeps the reagents at 4C. Author: Venkata S. Sabbisetti Page 5 of 11

6 Author: Venkata S. Sabbisetti Page 6 of 11

7 The Auto Sampler Unit (ASP) consists of a removable turntable, sample tube rack and sample barcode reader. The ASP accommodates 40 samples tubes. The carousel accommodates variety of tubes including 2 ml screw cap tubes and 1.8 ml eppdendorf tube. The ASP is equipped with barcode reader that can read various symbols including NW-7, Code 39, ITF, UPC and Code 128. Load the reagents in the reagent carousel and run inventory to record the inventory in the instrument. The inventory will provide the number of assays can be performed using the loaded reagents on the carousel. If a problem has occurred with the reagent a colour coded warning will be displayed as follows: Red - Insufficient reagent inventory Pink - Reagent expired Yellow - On-board stability period exceeded Insufficient Reagent Inventory and Reagent Expired conditions will remove the relevant test from the RUN MONITOR/TEST SELECT (F5) screen but on-board stability period exceeded conditions will allow the test to be performed with all results obtained flagged STB to indicate the stability period has been exceeded. Please note that expired reagents cannot be used on the analyser. Sample loading: Load the sample tubes directly into the carousel in the order 1-40 labeled on the carousel. Tubes should have 100 microliter volume when loaded into the carousel. Manual Test Selection Procedure 1. Select RUN MONITOR (F5) from the job menu. 2. Select TEST SELECT (F10) from the function key menu. 3. Move the cursor to the SAMPLE NUMBER field and click on either N (Normal Author: Venkata S. Sabbisetti Page 7 of 11

8 sample) or R (Replicate sample). Replicate sample is selected if the sample measurement is repeated (n=2) 5. The samples number will be automatically displayed. 6. A box will appear on screen prompting the user to ENTER SAMPLE INFORMATION DATA. Click OK if you wish to enter full patient demographic information or CANCEL if this option is not required 7. Press START (F1) to start analysis 8. A pop up window will appear to remind the user to perform water, waste and wash bottle checks. Click OK, The system will now begin analysis. 9. Analysis progress is displayed on the right of the RUN MONITOR/ RUN MONITOR(F9) screen. The sample status is displayed using a colour-coded system at each sample position on the left of the screen. The instrument status is indicated in the STATUS field on the top left corner of the RUN MONITOR screen 5. Data Generation & Processing 5.1 Data Output Select SYSTEM PARAMETERS/RESULTS (F12). The results screen will be displayed. Enter an * in all fileds, except sample type, to represent the wild card and display the results of all samples. Chick on the drop down menu of the RESULTS OUTPUT screen and select one of the following options: MONITOR (to view on screen) PRINTER (to print out results) FD (to save to flash drive) Click SEARCH. The screen will display a list of results for all samples. 6. Quality Control 6.1. Quality control of the Assay. Author: Venkata S. Sabbisetti Page 8 of 11

9 Quality control sample results can be viewed and searched by selecting CONTROL in the SAMPLE TYPE field in the System Parameters/Results (F12) screen. Quality controls sample results can be viewed and the information displayed as a Levy- Jennings chart. QC results can be stored for up to 6 months and can be backed up into hard drive. If neither of the assayed control results is within mean ± 2SD limits, then we will follow the westgard three-quality control rule and not accept the data. Author: Venkata S. Sabbisetti Page 9 of 11

10 6.2 Quality Control for each sample Sample measurements for will be repeated if the %CV of the sample is > 15% for any particular analyte. Sample measurements will be repeated if the instrument produces any run errors for a sample. If the values of the samples exceeds the range, the machine will do automatic resting at higher dilution. 6.3 Quality Control troubleshooting procedure If the controls fail the Westgard quality control procedure, then we will employ the following procedure to resolve the issue: Contact the Lab Director Rerun Control If the quality control values pass the above described westgard rules proceed with unknown testing If the quality control failed again, make up fresh reagents and rerun controls and document the problem If the quality control indicates that the westgard rules are passed, proceed with testing If QC fails again, recalibrate the assay (use new standard lot) If after calibration the QC determinations have passed westgard criteria, proceed with testing and document correction If Westgard criteria were not met after preparation of fresh reagents, do not proceed with the analysis and contact the Laboratory Director. 6.4 Data storage and reporting After the analysis has passed the quality control test Biomarker values of unknown samples with their corresponding %CV and SD will be copied and pasted into a master excel data workbook in the sequential order in sheet 1. Biomarker values, %CV, SD of quality control samples and proficiency samples will be copied and pasted in sheet 2. Both sheet 1 & 2 will be updated with addition of data from each run. The master data sheet reporting to cording center will contain the following information 1. Sample ID (Bio Con ID) 2. Date of the assay 3. Platform and the Instrument used for the assay 4. Reagent lot numbers 5. Concentration of Biomarker 6. % CV across the sample replicates 7. Biomarker levels, SD and % CV of each control sample with the description of each control sample Author: Venkata S. Sabbisetti 11 Page 10 of

11 8. Biomarker levels, SD and %CV of each proficiency samples with the description of each proficiency sample (the cells will be left blank if the proficiency samples are not run on that plate) Levey-Jennings plots of Control samples and proficiency samples (These plots will be updated and cumulative plots will be reported with each data submission. 6.5 Assay parameters Interference: The analytes below were tested up to the following levels and were found not to interfere at Creatinine concentrations of 60 mol/l and 260 mol/l The method is linear up to 122 mmol/l (375 mg/dl) of phosphorus and 225 mg/dl for creatinine. Creatinine Assay Inorganic Phosphorus Hemoglobin 750 mg/dl 250 mg/dl Free Bilirubin 15 mg/dl 25 mg/dl Conjugated Bilirubin 15 mg/dl 25 mg/dl Triglycerides 1000 mg/dl 250 mg/dl Glucose 300 mg/dl Not tested Ascorbate 3 mg/dl Not tested LLOD of creatinine in Plasma and Serum is 0.29 mg/dl and urine is 2.9 mg/dl LLOD of Phosphorus in Plasma is 0.39 mg/dl and urine is 2.73 mg/dl 7. References 1. Leaf DE, Jacob KA, Srivastava A, Sabbisetti VS, Martin A, Wolf M, Waikar SS. Fibroblast Growth Factor 23 levels Associate With AKI and Death in Critical Illness, JASN, 2017 Jun; 28 (6): Author: Venkata S. Sabbisetti 11 Page 11 of

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