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  1. 1. Basic Examination of Urine • examination of urine provides info regarding diseases of the kidney and lower urinary tract • clinical lab disciplines involved in urinalysis: chemistry, microbiology, routine wet urinalysis, cytology, and other specialty sections • new technologies include immunochemistry, molecular and genetic biology, DNA ploidy, and cell cycle analysis 2
  2. 2. 3 TYPES OF URINALYSIS 1. reagent strip (dipstick) urinalysis – for screening laboratories, physician offices, and patient home testing – remains a valuable frontline test for the early detection and monitoring of px for chemical abnormalities – encompasses the physiochemical analysis of urine – requires less sophisticated training of personnel – esily performed on multiple settings – provides accurate information in several clinical situations 3
  3. 3. 3 TYPES OF URINALYSIS 2. screening wet analysis • referred to as a routine or basic urinalysis • has 2 components: – macroscopic urinalysis or physiochemical determinations (appearance, sp gr, and multiparameter reagent strip measurements of several chemical constituents) – brightfield or phase-contrast examination of urine segment for evidence of hematuria, pyuria, cylinduria (casts), and crystalluria 4
  4. 4. 3 TYPES OF URINALYSIS 3. cytodiagnostic urinalysis • specialized urinalysis • cytologic approach to the urine sediment • more sensitive test pathologic test for evaluating urine sediment in several renal and lower urinary tract disorders • this specialized urine cytology test has replaced the quantitative Addis count method • provides sequential info regarding the progression or regression of many urinary system conditions 5
  5. 5. Types of urine specimens • random specimen – most commonly received specimen • First morning urine – the ideal screening specimen – used for routine screening, pregnancy tests and for determining orthostatic proteinuria • Fasting Specimen (Second Morning Specimen) – second voided urine specimen used for glucose monitoring • 24-hour specimen – timed specimen is used to determine concentration of a particular substance – Instruction: • Day 1 – 7am – patient voids and discards specimen – patient collects all urine for the next 24 hours • Day 2 – 7am – patient voids and adds this urine to the previously collected urine • Catheterized Specimen – used for bacterial culture • Midstream Clean-Catch Specimen – an ideal specimen for routine screening and for bacterial culture – patient is instructed to cleanse thoroughly the genitalia and is asked to collect the midstream portion of urine. When collecting, patient should be instructed to separate the labia in females or retract the foreskin in uncircumcised males. • Suprapubic Aspiration – used for bacterial culture and cytology sterile needle is introduced into the bladder to collect sample that is free of contaminants • Pediatric Specimen – soft, clear plastic bag w/ adhesive is attached to the genital portion 6
  6. 6. COMPONENTS OF BASIC (ROUTINE) URINALYSIS • specimen evaluation • gross/physical examination • chemical screening • sediment examination 7
  7. 7. Specimen Evaluation • urine specimen must be evaluated in terms of acceptability • Considerations: proper labeling, proper specimen for requested test, proper receptacle, storage conditions (time, temperature) and preservative, visible signs of contamination, and any transportation delays in moving the specimen to the lab • a properly labeled specimen must have the patient’s full name, and the date and time of collection 8
  8. 8. Gross/physical examination • Appearance and Color APPEARANCE CAUSE REMARKS Colorless Very dilute urine Polyuria, diabetes insipidus Cloudy Phosphates, carbonates, urates, uric acid Leukocytes, Red cells (“smoky”) bacteria, yeasts, spermatozoa, prostatic fluid, mucin, mucous threads, calculi, “gravel” clumps, pus, tissue, fecal contamination radiographic dye Phosphates, oxalates Rectovesical fistula In acid urine Milky Many neutrophils (pyuria) Fat Lipiduria,opalascent Chyluria, milky Emulsified paraffin Nephrosis, crush injury – soluble in ether Lymphatic obstruction – soluble in ether Vaginal creams Yellow Acriflavine Green Fluorescence 9
  9. 9. Yellow-orange Concentrated urine Urobilin in excess Bilirubin Dehydration, fever No yellow foam Yellow foam, if sufficient bilirubin Yellow-green Bilirubin-biliverdin Yellow foam Yellow-brown Bilirubin-biliverdin “beer” brown, yellow foam Red Hemoglobin Erythrocytes Myoglobin Porphyrin Fuscin, aniline dye Beets Menstrual contamination Positive reagent strip for blood Positive reagent strip for blood Positive reagent strip for blood May be colorless Foods, candy Yellow alkaline, genetic Clots, mucus 10
  10. 10. Red-purple Porphyrins May be colorless Red-brown Erythrocytes Hemoglobin on standing Methemoglobin Myoglobin Bilifuscin (dipyrrole) Acid pH Muscle injury Result of unstable hemoglobin Brown-black Methemoglobin Homogentisic acid Melanin Blood, acid pH On standing, alkaline On standing, rare Blue-green Indicans Pseudomonas infections Chlorophyll Small intestine infection Mouth deodorants 11
  11. 11. Color • Normal urine color – Yellow (due to urochrome) Color Pathologic NonPatho Drugs Orange Bilirubin Rhubarb Carrots Phenazopyridine Dark Yellow Bilirubin Urobilin Carrots Concentrated Fluorescein Green Oxidized bilirubin Biliverdin Pseudomonas Vit B complx Dithiazanine Nitorfurans Phenol Red or Pink RBCs Hemoglobin Myoglobin Porphyrins Beets Benzene Acetophenetidin Phenindione Brown or Black Biliary pigments Melanin Hmogentisic acid Rhubarb Phenol derivatives Methyldopa Levodopa Pale Yellow Diabetes mellitus Diabetes insipidus Large fluid intake Diuretics 12
  12. 12. Odor • usually not a part of routine urinalysis • ammoniacal odor of urine is due to breakdown of urea Odor Pathologic Nonpathologic Ammonia UTI Old urine Sweet Diabetes mellitus Starvation, dieting, strenuous exercise, vomiting, diarrhea Mousy Phenylketonuria Maple syrup Maple syrup disease Distinctive Garlic, onions, asparagus 13
  13. 13. Transparency (clarity) • Normal appearance: – freshly voided urine is usually clear • white cloudiness may be caused by precipitation of amorphous phosphates and carbonates • Causes of turbidity: – presence of phosphates, carbonates, crystals, red blood cells, leukocytes, epithelial cells, etc. 14
  14. 14. Specific Gravity • density of a substance compared with the density of a similar volume of distilled water at a similar temperature • used to assess kidney’s ability for reabsorption • Normal values = 1.015-1.025 15
  15. 15. ROUTINE URINALYSIS PROCEDURE Urine in specimen container Transfer urine in a clean tube Step 1 Physical Examination • note color & transparency • specific gravity & pH will be done using the Multistix (Step)2 Step 2 Chemical Examination • use Multistix Reagent Strip and test for glucose, bilirubin, ketones, specific gravity, blood, pH, protein, Urobilinogen, nitrite & leukocytes Centrifuge for 5 mins Decant supernatant & place a drop of the sediments onto a slide Step 3 Microscopic Examination • examine under LPO & HPO and write your findings in the result form 16
  16. 16. QUANTITATIVE DETERMINATION OF TOTAL PROTEIN IN URINE A. Pyrogallol Red-Molybdate Method Clinical Significance: • Urinary protein determinations are important in the evaluation of kidney function. The detection of protein in urine is considered evidence of renal disease and usually of glomerular disease. A minor evaluation of urinary albumin excretion from about 15-30 mg/day is now recognized to be a useful indicator of renal disease. Principle: • The Total Protein Test for Urine is based on the procedure developed by Watanabe et al which is a dye-binding colorimetric method utilizing pyrogallol red-molybdate complex. 17
  17. 17. • The pyrogallol red is combined w/ molybdenum acid, forming a red cplx w/ maximum abs at 467nm. When this cplx is combined w/ protein in acidic conditions, a blue-purple color develops w/ an inc in abs to 598nm. • Reagents: – PRM color reagent – pyrogallol red-molybdate soln buffered at pH 2.2 – Microprotein standard – (50 mg/dl) a soln of bovine serum albumin with preservative 18
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  20. 20. QUANTITATIVE DETERMINATION OF TOTAL PROTEIN IN URINE B. Microalbumin • An immunological, semi-quantitative determination of mcroalbuminuria up to 100 mg/L Principle: • Immunological detection of human albumin. The absorbed urine enters a zone on the strip containing a soluble antibody-gold-conjugate which specifically binds to urine albumin. Excess conjugate is retained in a separation zone containing immobilized human albumin so that only the albumin-loaded gold conjugate reaches the detection zone. The color produced (white to red) is directly related to the albumin content of the urine. Cross- reactions with other human proteins such as hemoglobin, transferring, Bence-Jones protein, α1-antitrypsin, acidic-α1-glycoprotein, α-amylase, Tamm-Horsfall protein and retinol-binding protein, as well as with IgG, IgA, human leukocytes and erythrocytes have been found to be <0.5%. Application: • For early detection and monitoring the course of incipient nephropathy in e.g. diabetics and hypertensive patients. Microalbuminuria denotes as albumin excretion of 20-200 mg/L.21
  21. 21. Test Component • One test strip contains: Monoclonal antibodies against human albumin (Immunoglobulin G) labeled with colloidal gold: 2.2ug, fixed albumin: 7.7 ug. Ideal Specimen: First morning urine Results: • Reaction colors lighter than the color block corresponding to approx 20 mg/L albumin indicate a physiological urine albumin conc. • The result is positive (i.e. indicates persistent microalbuminuria) when at least two of the three morning urines tested produce a reaction color corresponding to 20 mg/L (Threshold value of microalbuminuria) or more. 22
  22. 22. Determination of albumin concentrations above 100 mg/L • In order to determine albumin conc above 100 mg/L, the urine sample can be diluted e.g. by mixing 1 part of urine with 2 parts water. The original albumin concentration is then calculated by multiplying the result by 3. 23
  23. 23. COMBISTIX / MULTISTIX Multiple Reagent Strips for Urinalysis • Semi-quantitative determination of the following parameters: – Specific gravity – Bilirubin – pH – Hemoglobin/Myoglobin – Albumin – Urobilinogen – Glucose – Nitrite – Ketone – Esterase Leucocytes Nitrite Urobilinogen Protein pH Blood SpecificGravity Ketone Bilirubin Glucose 24
  24. 24. Ketone (15 secs) • Principle: Sodium Nitroprusside Rxn • The test is based on the development of colors ranging from buff pink for negative reading to maroon when acetoacetc acid reacts with nitro-prusside. Normally no ketones are present in urine. As little as 0.5-1 mmol/L acetoacetic acid is detectable. Positive results (trace or less) may occur with highly pigmented urine specimens or those containing large amounts of L-Dopa metabolites. • Reagent Composition: Sodium nitroprusside 25
  25. 25. Bilirubin (20 secs) • Principle: Diazo reaction Coupling of bilirubin w/ diazotized dichloroaniline in a strongly acid medium yielding various shades of tan. Reagent Composition: 2,4 dichloroaniline diazonium salt • This test is based on the coupling of biilrubin w/ diazotized dichloroaniline in a strongly acid medium yielding various shades of tan. Normally, no bilirubin is detectable in urine by even the most sensitive tests, so any positive result calls for further investigation. The test has a sensitivity of 7-14 umol/L bilirubin. Colors that do not match the color blocks may result from the presence of bile pigments other than bilirubin, indicating bile pigment abnormalities and suggesting the need for further testing with ICTOTEST reagent tablet, which are 2 to 4 times more sensitive than the strip test. 26
  26. 26. Glucose (30 secs for quantitative results) Principle: Glucose oxidase, double sequential enzyme rxn • Glucose oxidase converts glucose to gluconic acid and hydrogen peroxide. Peroxidase then catalyzes the rxn of hydrogen peroxide w/ a potassium iodide (KI) chromogen to form a green to brown color. Reagent Composition: Glucose oxidase, peroxidase, potassium iodide, buffer, non-reactive ingredients. • In a double sequential enzyme rxn, glucose oxidase converts glucose to gluconic acid and hydrogen peroxide. Peroxidase then catalyzes the rxn of H2O2 w/ KI chromogen to form a green to brown color. This test yields negative results w/ normal urines. Significant abnormality may be indicated by results of as little as 5 mmol/L if found consistently 27
  27. 27. Blood (40 seconds) Principle: Peroxidase-like activity of hemoglobin Catalyzes the reaction of cumene hydroperoxide and tetramethylybenzidine Note: the appearance of green spots on the reacted reagent area indicates the presence of intact red cells in the urine. Reagent Composition: Cumene hydroperoxide, tetramethylbenzidine This test is based on the peroxide-like activity of hemoglobin w/c catalyzes the rxn of cumene hydroperoxide and 3,3’, 5,5’- tetramethylbenzidine. The resulting color ranges from orange through green to dark blue. 28
  28. 28. Urobilinogen (45 seconds) Principle: Ehrlich reaction p-dimethly-amino-benzaldehyde reacts w/ urobilinogen in a strongly acid medium to form a brown orange color. Reagent Composition: Para-dimethylaminobenzaldehyde This test is based on the Ehrlich reaction in which p- dimethyl-amino-benzaldehyde reacts w/ urobilinogen in a strongly acid medium to form a brown orange color. In healthy population, the normal urine urobilinogen range is 1.6-16 umol/L urobilinogen. 29
  29. 29. Specific Gravity (45-60 seconds) Principle: This test is based upon the ionic conc of urine. The pKa’s of certain pretreated polyelectrolytes change in relation to the ionic conc of the urine. In the presence of an indicator, colors range from deep blue-green in urine of low ionic conc through green and yellow- green in urines of increasing ionic conc. Reagent Composition: Bromthymol blue, poly (methyl vinyl ether maleic anhydride), sodium hydroxide 30
  30. 30. pH Principle: Double indicator system Gives a range of colors from orange through yellow and green to blue and permits differentiation to w/in 0.5 pH units in the range pH 5 to pH 8.5. Reagent Composition: methyl red, bromthymol blue, nonreactive ingredients This test is based on the double indicator system principle. Readings are not affected by variations in urinary buffer concentration, but contamination by reagent washed from an overwetted adjacent reagent area may affect results. 31
  31. 31. Protein Principle: Protein-error-of-indicators The presence of protein results in development of a green color Reagent Composition: Tetrabromphenol blue, buffer, non-reactive ingredients. The test is based on the protein-error-of-indicators principle. The presence of protein results in development of a green color (echo!). The test yields negative results w/ normal urines, so any positive result greater than “Trace” indicates significant proteinuria. Clinical judgment may be used to interpret “Trace” results, w/c may occur w/ urines of high specific gravity w/ non-significant protein-uria. The “trace” result corresponds to 0.05-0.2 g/L albumin, but the test is less sensitive to globulin, Bence-Jones protein and mucoprotein, so a negative result does not rule out the presence of these proteins. 32
  32. 32. Nitrite (60 seconds) Principle: Greiss reaction Conversion of nitrate (derived from diet) to nitrite by the action of principally Gram negative bacteria in the urine Reagent Composition: p-arsanilic acid, tetrahydrobenzo(h)-quinolin- 3-ol This test depends upon the conversion of nitrate (derived from diet) to nitrite by the action of principally Gram negative bacteria in the urine. The test is specific for nitrite and will not react w/ any other substance normally excreted in urine. Pink spots or pink edges should not be interpreted as positive result 33
  33. 33. Leukocytes (2 minutes) Principle: Granulocytic leukocytes contain esterases that catalyze the hydrolysis of the derivatized pyrrole amino acid ester to liberate 3-hydroxy-5-phenyl pyrrole. This pyrrole then reacts w/ a diazonium salt to produce a purple product. Reagent Composition: Derivatized pyrrole amino acid ester, diazonium salt 34
  34. 34. MICROSCOPIC ANALYSIS OF URINE •Fresh or adequately preserved urine specimen (approximately 10-15 m) is placed preferably on a conical tube and centrifuged for 5 minutes. •Decant supernatant and the sediment is resuspended with the remaining urine in the tube (usually 0.5 mL or 1.0 mL) •Using a pipet, place a drop of resuspended sediment on a clean slide and cover with a cover slip •Examine the sediment first using the LPO •Shift to HPO to identify specific types of cells, casts, bacteria and crystals 35
  35. 35. Manner of Reporting: 36 Read under LPO Casts Hyalin Granular Waxy Pus cell RBC Cells Squamous cells Renal cells Mucus threads Amorphous urates Uric acid Calcium oxalate Amorphous PO4 Triple PO4 Report As # of casts seen/ cover slip Few, occasional, moderate or many OR +, ++, +++, ++++ Few, occasional, moderate or many OR +, ++, +++, ++++ Read under HPO RBC WBC Average of RBC/WBC seen in 10 fields/HPF Yeast cells Bacteria Few, occasional, moderate or many OR +, ++, +++, ++++
  36. 36. Manner of Reporting: Elements Report As RBCs and WBCs Average of RBC or WBC seen in 10 high power fields (HPF) Casts Average casts seen per cover slip ( __/cs) Epithelial cells Report as: few, occasional, moderate, many or +,++,+++,++++ Other elements (Bacteria, yeasts) Report as: few, occasional, moderate, many or +,++,+++,++++ 37
  38. 38. URINARY SEDIMENTS • 1. Cells • cells present in urinary sediment include WBCs, RBCs and epithelial cells. These cells can be anywhere in the urinary tract from the tubules to the urethra. 39
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  41. 41. • 2. Casts • Casts are cylindric structures formed primarily within the lumen of the distal convoluted tubule and collecting duct. The major constituent of casts is Tamm-Horsfall protein, a glycoprotein excreted by the renal tubular cells. 42
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  44. 44. • 3. Microorganisms and Parasites 3. Microorganisms and Parasites 45
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  46. 46. • 4. Crystals • Crystals are frequently found in the urine. They are formed by the precipitation of urine salts subjected o changes in pH, temperature or concentration, which affect their solubility. Crystals are identified by their appearance, solubility and pH. 47
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