References Required document for Laboratory Accreditation by the College of American Pathologists.

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1 Subject Urinalysis Clinitek Advantus Regional Index Number Lab-1589 Section Laboratory Subsection Regional Clinic / Affiliate Hospital Laboratories Category Departmental Contact Elizabeth A Kamprud Last Revised 5/10/2017 References Required document for Laboratory Accreditation by the College of American Pathologists. Applicable To Employees of the Gundersen Health Systems laboratories. Detail CLINICAL SIGNIFICANCE: The urinalysis is a composite examination that is usually carried out with a single specimen of urine performed and recorded in a systematic sequence. This examination offers many invaluable clues in the detection, differential diagnosis and evaluation of urinary tract disorders, especially in the areas of metabolic and renal disorders. PRINCIPLE: Routine urinalysis consists of a specific gravity, chemical examination, including: color, clarity, ph, albumin, glucose, ketones, bile, leukocyte esterase, nitrite and blood. Microscopic examination, if needed, includes identification of cells, casts, crystals, bacteria and miscellaneous. The Clinitek Advantus Urinalysis analyzer is a semi-automated, bench-top analyzer designed to read urinalysis reagent strips. The analyzer is a reflectance spectrophotometer that analyzes the color and intensity of the light reflected from the reagent area and reports the results in clinically meaningful units. Principles of the individual dipstick tests are found under data sheets in Non-Automated Procedure Manual. SPECIMEN: cc of clean voided, catheterized or random urine (first voided morning specimen is the most concentrated) in a labeled container. The minimum specimen volume is 1.0 cc. Turbid specimens containing amorphous urates should be heated under hot tap water to dissolve the crystals. 2. Specimen should be refrigerated at 2-8 C, if it is not run immediately. Urines ideally should be tested within 30 minutes, but up to 2 hours at room temperature is acceptable. Urines can be refrigerated for up to 24 hours without gross alterations. 3. Unacceptable specimens: Specimens kept at room temperature for more than 2 hours: Fecal contamination: Improperly labeled specimens. Specimens that are over 24 hours old old. 4. An aliquot is saved for 48 hours for additional testing as needed for other areas of the lab. REAGENTS/MATERIALS: 1. Multistik 10 SG, Bayer3. Lab-1589 Urinalysis Clinitek Advantus Regional Page 1 of 9

2 a. Store strips at room temperature. b. Good until expiration date stated on package. c. Do not remove desiccant from bottle or store in direct sunlight. 2. Whale tubes. Fisherbrand Urisystem Centrifuge tubes. 3. Plain glass slides, Fisherbrand A 4. Plastic pipettes, Fisherbrand Coverslips, 22x22, Fisherbrand B EQUIPMENT INSTRUMENTATION: Calibration: Calibration is performed at each readhead immediately before each reagent strip is read. The fixed platform contains 2 white calibration bars, positioned directly under each readhead. As a strip comes into position under a readhead, the analyzer reads the calibration bar and calibrates for that scanning cycle. The analyzer then scans the reagent strip and stores the data in memory. Maintenance: 1. Clinitek Advantus daily maintenance is documented on the Urinalysis Maintenance. For daily cleaning, see Clinitek Advantus Operators manual pg Fixed head centrifuge-clean weekly. Clean centrifuge shell with Oxivir. Refer to Lab-0105 if breakage or spill occurs. 3. Microscope- phase. Phase alignment is checked weekly. Refer to 'Repair Record, Phase Alignment, Atlas of Sediment' notebook. QUALITY CONTROL: BioRad quantify Plus Control - Level 1 BioRad quantify Plus Control- Level 2 1. Preparation: Mix controls. Pour a 5 ml aliquot into a Whale tube. Return bottles to refrigerator. Warm aliquot for 15 minutes. Test aliquot on Clinitek Advantus (macroscopic), then spin and test as a patient specimen (microscopic). 2. Storage & Stability. a. Store vials at 2-8 C until the expiration date on the label. b. Once opened bottles are stable for 31 days at 2-30 C. The bottle is dated when opened and the new opened expiration date added. c. QC is run every 24 hours and documented in the lab computer. d. Go to Lab Outstanding List to verify the macroscopic portion. The microscopic QC will auto order. e. New lot controls are run in parallel for a minimum of 15 data points from which new limits are established. The new limits are entered into the lab computer. f. If the QC is not within the set limits, the computer will generate an action log and the tech must enter a response. If QC fails, rerun. If QC fails again, test with fresh QC material. If QC fails again, see troubleshooting section of the operators manual. Patients will not be resulted until QC is within set limits g. All QC is reviewed monthly by the Supervisor. h. Every new lot number of MULTISTIX must have QC done before patients are tested. i. Test new lot number of MULTISTIX against the current lot# of QC. Lab-1589 Urinalysis Clinitek Advantus Regional Page 2 of 9

3 ii. QC limits rarely change, however if there is a change the new QC limits are entered into the lab computer. iii. If new lot number fails QC, retest. If still not acceptable, test fresh QC material. If QC fails on fresh material, the new multistix lot is rejected. iv. QC is verified in the lab computer. v. Refer to Lab Implementation PROCEDURE: Routine Urinalysis Macroscopic 1. Test fresh urine or urine in a preservative tube. If urine has been refrigerated, allow to warm to room temperature. All urines are done as soon as they arrive and should not be routinely put in the refrigerator. 2. Program the instrument for patients to be loaded. a. Press ID key from the Ready/Run screen. b. Enter the sample number using the scanner. c. Press the enter key. d. When all urines have been entered into the load list, press the up arrow. The Ready/Run screen will show each ID number, check that the ID Number & specimen match. 3. Completely immerse all reagent areas of the strip into fresh, well-mixed, uncentrifuged urine. Immediately remove the strip. While removing, slowly run the edge of the entire length of the strip against the side of the urine container to remove excess urine. Do not blot the edge of the strip against a paper towel-results may differ if strips are blotted. 4. Place the reagent strip, with reagent areas facing up, onto the strip supports of the strip leading station, to the right of the small embossed arrow. The strip should be lying parallel to the surface of the platform and the end of the strip should be against the rear wall of the platform. 5. The presence of the strip is detected as soon as it is placed on the loading station and the timing and movement functions are activated. 6. The next number will now appear on the instrument and the second sample may be tested. 7. When the strip for the last load listed specimen has been moved to the read area, you will not be allowed to place any additional strips on the table. The push bar will stay at the right side and the display will change to "Completing Run, Please Wait." 8. Results will cross the LIS interface. A microscopic will be reflexed if it meets one or more of the following criteria. a. Color abnormal (red, amber, green, brown and orange) b. Clarity turbid c. Leukocytes positive d. Nitrite positive e. Protein 1+, 2+, 3+ f. Blood trace, 1+, 2+, 3+ Urinalysis Microscopic: 1. Centrifuge specimen for 5 minutes at g. (Set at 1.8 on centrifuge) in a Whale tube. 2. Microscopic results are based on a volume of cc of specimen. When the volume is less than 10 cc, document the volume of specimen. Lab-1589 Urinalysis Clinitek Advantus Regional Page 3 of 9

4 3. After spinning, decant supernatant by tipping tube. DO NOT SHAKE. Approximately 0.4 cc should remain in tube. 4. Mix sediment well and holding the pipette upright, place a free-falling drop on slide. Coverslip the sediment with a 22X22 mm coverslip. Use one patient per slide to avoid contamination. 5. Examine sediment under low power screening edges of coverslip for casts and large elements. Then examine sediment under high power. 6. Enumerate and report all cells/elements under high power. 7. Microscopic elements in an extremely cellular sediment can be identified by using a large coverslip (22X40 mm) to spread the sample thinner, multiple the results by 2 to compensate for the larger coverslip. A 1:2 dilution can also be made using saline and the results multiplied by two. 8. Report results in LIS. Reporting Results: 1. Glucose, protein, and ketones are reported semi-quantitatively as trace, 1+, 2+, or Bilirubin is reported as, 1+, 2+, or Leukocytes are reported semi-quantitatively as trace, 1+, 2+, or Urobilinogen is not reported. 5. ph is reported in quantitative ph units. 6. Blood is reported semi-quantitatively as trace, 1+, 2+, or Nitrate is reported positive or negative. 8. Specific Gravity is reported qualitatively, ranging from to in 0.05 increments. 9. Clarity is reported quantitatively as clear, slightly cloudy, cloudy, turbid or other. 10. Color is reported as colorless, yellow, orange, brown, red, blue, green and other. 11. RBC s and WBCs are reported as cells per HPF up to 100. Counts over 100 will be reported as > WBC clumps are reported semi-quantitatively per HPF as occ, few, moderate or many.* 13. Renal and transitional are reported in the category Non Squamous per HPF as occ, few, moderate or many. * 14. Squamous epithelial cells are reported per HPF as occ, few, mod or many. * 15. Bacteria are reported per HPF as <10, or > Crystals are reported per HPF as rare, occ, few, moderate or many. * 17. All casts are reported, by type per HPF as <1, 1-10, > Yeast is reported as present. 19. Sperm is reported as present. 20. Fat is reported as present. 21. Mucous and clue cells will not be reported. *The definitions of the semi-quantitative terms that apply to WBC clumps, squamous epith, nonsquamous epithelial and crystals are as follows: Occasional 0-2/ HPF Few 2-5/ HPF Moderate 5-10/HPF Many > 10/ HPF PROCEDURE NOTES: Lab-1589 Urinalysis Clinitek Advantus Regional Page 4 of 9

5 Orange urine: The color orange as a result will automatically cancel out all the macroscopic tests and reflex a urine microscopic. An orange urine will leave the reagent strip discolored, while an amber urine will not leave any discoloration on the strip. Do a visual check. Do not dip the urine. Manually enter the color and clarity into LIS. Save. This will reflex the comment Unable to report ph, Specific Gravity, Albumin, Glucose, Ketone, Bilirubin, Leukocyte Esterase and Nitrates due to interfering Substances. Run the microscopic and after both the macroscopic and microscopic are resulted, then verify results. A microscopic will be done if one or more of the following are reported on the macroscopic: 1. Color abnormal (red, amber, green, brown and orange) 2. Clarity turbid 3. Leukocytes positive 4. Nitrite positive 5. Protein Blood trace, Grossly bloody urines are handled as follows: 1. Spin the urine and dip the supernatant. 2. If the supernatant is pink tinged or yellow, the occult blood should be resulted as 3+ and the other parameters are reported. Change color to red and clarity to turbid. 3. If the supernatant is still red, use the comment.beakuagrosslybloody- unable to result, for glucose, ketones, albumin, nitrite, bilirubin, ph and leukocytes. The blood is resulted as 3+ and a refractometer specific gravity is resulted. Specimens requiring refractometer specific gravity must be sent to La Crosse campus for the refractometer specific gravity. 4. You can final verify but not prelim verify. If reports were given to the medical staff verification should occur. You can leave the SG blank. These will be resulted and verified in LAX. Enter a note in the lab comments (yellow Box) saying, SG sent to lax this will at least provide a method type for the test (CAP reg) i. You cannot put this on a packing list so, you must send to lax and call and alert the Urinalysis staff (52215) to let folks know its coming. Correlation of microscopic results with the chemistry results: Correlate microscopic findings of casts, bacteria, WBCs and RBCs with strip results of positive protein (look for casts); nitrite (look for bacteria), leukocytes (look for WBCs) and occult blood (look for red cells). If occult blood is negative and RBCs are found microscopically, ascorbic acid interference should be suspected. Be sure the formed elements are RBCs, not yeast or unusual Ca oxalate crystal forms. If the occult blood is positive but no RBCs are seen microscopically, check for low specific gravity indicating a hypotonic solution which may lyse RBCs. Lab-1589 Urinalysis Clinitek Advantus Regional Page 5 of 9

6 If the Leukocyte esterase is negative and 5 or more WBC/ HPF are seen, check for high specific gravity. A hypertonic solution may cause WBCs to crenate, thereby preventing the WBC granules from reacting with the test pad. If the Leukocyte esterase is positive and less than 5 WBC/HPF are seen, check for low specific gravity. A hypotonic solution may cause the WBCs to lyse. Large numbers of eosinophils or Trichomonas Vaginalis may provide sufficient esterase to give a positive result. If fat globules are suspected, examine with polarized microscope. If polarizing the specimen is not definitive, perform a Sudan Fat Stain (see Lab-8160 Sudan Fat Stain). Increased number of abnormal casts must be accompanied by proteinuria, also the degree of proteinuria can vary. In contrast, proteinuria may occur without cast formation. 1. Hyaline Casts: Hyaline casts are the result of solidification of Tamm-Horsfall mucoprotein, which is secreted by the renal tubular cells, and they may be present without significant proteinuria. A few may be seen in the urine of a healthy person. They may be seen in increased numbers after strenuous exercise and in some renal disease. 2. WBC casts: A WBC casts are present in noninfectious inflammations and in renal infections. They can be seen in acute pyelonephritis, interstitial nephritis, and lupus nephritis. They can also be present in glomerular disease. 3. Mixed cellular casts: A cellular cast may be composed of any of the cells found in the urine sediment such as RBC, WBC and epithelial cell. Bacterial cells casts have also been described. 4. RBC casts: RBCs may be found in a cast either as the result of leakage of RBCs through the glomerular membrane or be bleeding into the tubules at any point along the nephron. Other forms of RBC cast include hemoglobin cast, which are the result of degeneration of RBCs with the cast matrix. The cells are no longer visible, yet the remaining hemoglobin pigment imparts a characteristic orange-yellow or red-brown color, which distinguishes them from waxy cast. Dipstick should have positive blood and protein. 5. Epithelial cell cast: Usually composed of renal epithelial cells. A serious pathologic finding, associate with acute tubular necrosis, viral disease such as infection with cytomegalovirus, and exposure to nephrotoxic substances such as mercury, ethylene glycol and various drugs. They may be difficult to distinguish from WBC cast especially as they begin to degenerate into granular cast, because of this, report with caution. 6. Granular Casts: the result of degeneration of cells in the cellular casts. Their significance lies with the cast from which they were formed. Granular cast may also be seen following periods of stress and strenuous exercise. No distinction will be made between coarse and fine granular. 7. Waxy Cast: Represent the final stage in the degeneration of cells within a cast from cellular to granular to waxy. Associated with severe chronic renal disease and renal amyloidosis, waxy casts are only rarely seen in acute renal disease. Waxy casts are homogeneous, like hyaline casts, but they are more refractile, with sharper outlines and they tend to have broken or blunt ends and fissure or cracks along the sides. It is important that waxy cast not be confused with fibers from disposable diapers or other contaminants. The presence of protein on the reagent strip and lack of polarization of the waxy cast with polarized light will help with this distinction. Lab-1589 Urinalysis Clinitek Advantus Regional Page 6 of 9

7 8. Fatty Casts: Contain globules of fat, either as triglyceride or normal fat, which can be stained with Sudan Stain, or as cholesterol, which will polarize as a Maltese cross. Nitrite a positive nitrite is a good indicator of the presence of bacteria. nitrite does not necessarily indicate the absence of bacteria. A negative nitrite does not necessarily indicate the absence of bacteria. Bacteria can be found in urine with a negative nitrite if the urine lacked the necessary four-hour bladder incubation to yield a positive nitrite or if the bacterial are of the non-nitrite reducing types. Crystal formation is dependent on urine ph. Refer to reference material for identification. AMR: is measured from 5.0 to 9.0 in 0.5 increments. Specific Gravity is measured from to in 0.05 increments. Protein is measured as negative, trace, 1+, 2+, 3+. Sensitivity is mg/dl albumin. Glucose is measured as negative, trace, 1+, 2+, 3+. Sensitivity is mg/dl. Occult blood is measured as negative, trace, 1+, 2+, 3+. Sensitivity is mg/dl Hgb. Leukocyte esterase is measured as negative, trace, 1+, 2+, 3+. Sensitivity is 5-15 WBC/HPF. Ketones are measured as negative, trace, 1+, 2+, 3+. Sensitivity is 5-10 mg/dl acetoacetic acid. Bilirubin is measured as negative, 1+, 2+, and 3+. Sensitivity is mg/dl. Nitrite is measured as positive or negative. Sensitivity is mg/dl nitrite ion. Urobilinogen is not reported. Clarity is reported as clear, slightly cloudy, cloudy, turbid, or other. Color is reported as yellow, orange, red, green, blue, brown or other. CALCULATIONS: N/A INTERPRETATION: Chemistry Tests Normal Ranges Microscopy Elements Normal Ranges ph WBC 0-5 /HPF Specific Gravity RBC 0-2/HPF Leukocyte Esterase Bacteria <10/HPF Protein, Qualitative Occult Blood Nitrite Glucose Ketone Bilirubin Color Yellow, light yellow, colorless Clarity Clear, slightly cloudy LIMITATIONS: Urinalysis - macroscopic 1. The Specific Gravity is measured by spectrophotometry. 2. Results reported by the analyzer may be different from the color seen visually. This is due to the inherent differences between the human eye and the optical system of the analyzer. Report the visual results. Edit results on interface prior to verification. 3. See chart for common causes of false positive & negative results: Lab-1589 Urinalysis Clinitek Advantus Regional Page 7 of 9

8 ANALYTE Glucose CAUSES OF FALSE NEGATIVE RESULTS Increased amounts of ascorbic acid CAUSES OF FALSE POSITIVE RESULTS Presence of oxidizing substances such as chlorine or hypochlorite, ph <4.0. Reducing substances* Protein Urine with ph <3.0 Urine with large amount of Hgb, ph >8.0, contrast medium, disinfectants including quaternary ammonium compounds. The lab computer will reflex a comment, Albumin assay may not be accurate do to the high ph. Bilirubin* Occult Blood Ascorbic acid, uric acid and nitrites. Urine with elevated specific gravity, protein or ascorbic acid. Presence of urobilinogen, Etodiac. Presence of oxidizing substances such as chlorine or hypochlorite. Ketones N/A Drugs such as L-Dopa, BSP, PSP, Phenylketone, Cephalosporine, Aldose, Reductive antienzyme. Nitrites Urine with elevated specific gravity, or ascorbic acid. Leukocytes Urine with glucose >500, protein >300, ph 5.0 or less, elevated specific gravity. N/A ph*** N/A N/A Formaldehyde *If screening for galactosemia is desired, a urine specimen should be submitted for an urine galactose. If a request for urine reducing substances is made by a clinician, contact ordering provider to confirm that urine galactose testing is appropriate. **Unstable at room temperature and in light. ***Old urine increases in alkalinity. 4. See the Multistix product insert for expanded list of limitations for each test. These test strips rely on a range of color changes to measure the concentration of the various analytes. Substances that cause abnormal urine color, such as drugs containing azo dyes, nitrofurantoin and riboflavin, may affect the readability of the test strips. 5. Manual entry of patient identification is available for computer downtimes. See Lab-0646 Urinalysis-Microscopic: 1. Grossly bloody specimens may require the addition of 2% glacial acetic acid (2 ml glacial acetic acid: 98 ml DI water) to identify the presence of WBCs. Add Lab-1589 Urinalysis Clinitek Advantus Regional Page 8 of 9

9 one drop of 2% glacial acetic acid to one drop of sediment, coverslip and scan for WBC nuclear remnants. 2. Normal values were determined on a ml concentration of specimen. When performing the microscopic on < 10ml, document the volume in LIS. When performing the microscopic on <1ml, attach the comment <1cc unspun microscopic performed. REVIEW & 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 Supervisor, Manager, Technical Leader, Medical Director or designated person. Changes require retyping document or form and review by the Medical Director. REFERENCE: 1. Clinical Laboratory Technical Procedure Manuals, NCCLS, and Ed, Approved Guidelines, July 1992, Vol 12, No Urinalysis and collection, Transportation, and Preservation of Urine Specimens; Approved Guidelines, NCCLS< GP16A, Vol. 15, No Urinalysis and Body Fluids, Ringsrud and Linne. 4. Fundamentals of Urine and Body Fluid Analysis, Brunzel, Nancy, WB Saunders & Co, Clinitek Advantus Operators manual. Lab-1589 Urinalysis Clinitek Advantus Regional Page 9 of 9