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Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Chapter 4: Chemical Analysis of Urine Graff’s Textbook of Urinalysis and Body Fluids,

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Presentation on theme: "Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Chapter 4: Chemical Analysis of Urine Graff’s Textbook of Urinalysis and Body Fluids,"— Presentation transcript:

1 Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Chapter 4: Chemical Analysis of Urine Graff’s Textbook of Urinalysis and Body Fluids, Second Edition Mundt, L. & Shanahan K.

2 Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Chapter Outline Introduction to reagent strip testing pH Protein Glucose Ketones Blood Bilirubin Urobilinogen Nitrite Leukocyte esterase Additional parameters

3 Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Introduction to reagent strip testing Illustration of Multistix 10 SG

4 Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Introduction to reagent strip testing (cont.) Dipping method

5 Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Introduction to reagent strip testing (cont.) Reading method

6 Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Introduction to reagent strip testing (cont.) For accurate results: –Store reagent strips in their bottle with lid closed at room temperature. –Do not expose strips to Sunlight Heat Cold Volatile substances Moisture (each bottle contains a desiccant) –Only remove the number of strips you need for immediate testing. –Do not use strips that have discolored pads. –Do not use expired reagent strips. –Test urine which is brought to room temperature.

7 Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Introduction to reagent strip testing (cont.)

8 Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Urine pH Kidneys regulate acid-base balance pH changes demonstrate and assess this function Must be evaluated in clinical context

9 Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Urine pH Acid indicator – methyl red Alkaline indicator – bromthymol blue Read 60 seconds after dipping pH ranges from 5.0 to 8.5 in half units pH color chart

10 Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Urine pH pH

11 Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Urine pH: Significance Persistent Acidity –Acidifying drugs –Dehydration –Diabetes –Diarrhea –Fever –Gout –High protein diets –Pulmonary emphysema

12 Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Urine pH: Significance (cont.) Persistent alkalinity –Alkaline drugs –Acute and chronic renal failure –Diuretics –Renal tubular acidosis –Urinary tract infections

13 Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Urine protein Small amounts of low–molecular weight protein are filtered at the glomerulus Most of this protein is reabsorbed in the tubules Less than 150 mg/24 h (or 20mg/dL) is excreted Mucoprotein Tamm–Horsfall is secreted by the renal tubules is also excreted

14 Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Urine protein (cont.) Protein color chart Principle: “protein error of indicators” Tetrabromphenol blue most common indicator Color ranges from yellow to blue Detects primarily albumin Negative does not rule out other significant proteins

15 Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Urine protein: false positive Highly buffered alkaline urine (medications or old urine) Prolonged exposure to the sample Container cleaning compounds (quaternary ammonia) Phenazopyridine Plasma expander polyvinylpyrrolidon Chlorhexidine gluconate (skin cleansers) Blood

16 Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Urine protein: false negative Dilute urines Elevated amounts of proteins other than albumin

17 Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Urine protein: significance Physiology causes Exercise Emotional stress Exposure to heat or cold Fever Pregnancy

18 Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Urine protein: significance Pathologic causes Glomerular nephritis Pyelonephritis Malignant hypertension

19 Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Urine glucose Normally contained in glomerular ultrafiltrate Reabsorbed in the proximal tubule Usually not present unless threshold levels exceeded 160-180 mg/dl

20 Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Urine glucose Glucose color chart Reaction A: glucose oxidase Glucose + O2 gluconic acid + H2O2 Reaction B: peroxidase H2O2 + chromogen oxidized chromogen

21 Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Urine glucose: false positive Oxidizing cleaning agents Peroxide Hypochorite Elevated urobiliongen with some automated methods

22 Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Urine glucose: false negative Decreased sensitivity in: Cool urine Urine with high specific gravity Alkaline urine High ketone levels Ascorbic acid (Vitamin C) high doses

23 Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Urine with reducing substances Other sugars Fructose Galactose Lactose Maltose Dextrins Homogentisic acid Glucuronates

24 Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Clinitest Clinitest color chart

25 Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Clinitest: false positive Ascorbic acid Nalidixic acid Cephalosporins Probenecid Urinary preservatives: –formalin –formaldehyde

26 Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Clinitest: false negative Technique errors

27 Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Urine glucose: significance Normal renal threshold – 160-189 mg/dl Glycosuria dependent on: Blood glucose levels Glomerular filtration rate Tubular reabsorption Seen in diabetes and congenital forms of glucosuria

28 Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Urine reducing sugars: significance Routinely performed on urine from children 2 years old and younger Early detection of galactosemia

29 Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Urine ketones: normal formation Ketones result from the breakdown of fat. One of the intermediate products of fatty acid Breakdown is acetyl CoA. Acetyl CoA enters the citric acid cycle (Krebs cycle) in the body if fat and carbohydrate degradation are appropriately balanced. The first step in the Krebs cycle is the reaction of acetyl CoA with oxaloacetate to yield citrate.

30 Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Urine ketones: excessive formation Oxaloacetate will be used to form glucose when carbohydrates are absent or improperly used. No oxaloacetate available for condensation with acetyl CoA. CoA cannot enter the Krebs cycle and is diverted to the formation of ketone bodies.

31 Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Urine ketones O || CO 2 CH 3 -C-CH 3 O (Acetone) || H 3 C-C-CH 2 -COOH (Acetoacetic acid) || NADH 2 CH 3 -CH-CH 2 -COOH (β-hydroxybutyric acid) NAD + OH

32 Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Urine ketones Ketone color chart alkaline pH Acetoacetic acid + Na nitroprusside + Glycine violet–purple color

33 Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Urine ketone: false positive or atypical color Highly pigmented urines Combination of high specific gravity and a low pH Levodopa metabolites Sulfhydryl groups Phenylketones Phthalein compounds Positive and questionable results may be confirmed with a tablet test.

34 Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Urine ketone: false negative Controls solutions that use acetone.

35 Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Urine ketones confirmatory test Acetest color chart

36 Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Urine ketone Ketone blood level are normally very low (2-4 mg/dl). –20% acetoacetic acid –2% acetone –78% B-hydroxybutyric acid. Acetone is lost into the air at room temperature

37 Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Urine ketone: significance Diabetes mellitus Diarrhea Exposure to cold Fasting Fever Insufficient carbohydrate intake Malnutrition Strenuous exercise Vomiting

38 Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Urine blood/hemoglobin Hematuria Glomerulus Renal tubules Ureters Bladder Hemoglobinuria Glomerular filtrate Myoglobinuria Glomerular filtrate

39 Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Urine blood/hemoglobin Blood color chart hemoglobin H 2 O 2 + chromogen oxidized chromogen+ H 2 O 2 peroxide

40 Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Urine blood/hemoglobin: false positive Oxidizing contaminants Hypochlorites Bacterial peroxidases Menstrual blood Povidoneiodine (Betadine)

41 Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Urine blood/hemoglobin: false negative Technique errors inadequate mixing Formalin preservative High concentrations of ascorbic acid (Vitamin C) Captoprin (Capoten) High specific gravity Nitrites Proteins

42 Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Urine blood/hemoglobin: significance Hematuria Hemoglobinuria Myoglobinuria

43 Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Urine blood/hemoglobin: hematuria Intact RBCs If lysed in urine, “ghosts” may be present in sediment Urine may appear normal with small amounts Urine is red with greater amounts of blood

44 Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Urine blood/hemoglobin: hematuria Can indicate renal diseases Glomerular Tubular Interstitial Vascular Also present in patients with Lithiasis (kidney stones) Urinary tract infections Urinary tract tumors

45 Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Urine blood/hemoglobin: hemoglobinuria Free hemoglobin Intervascular hemolysis Incompatible blood transfusions Hemolytic anemia Burns Cold auto-immune anemia Electric shock Lysis only in urine does not carry the same significance

46 Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Urine blood/hemoglobin: myoglobinuria Damage to cardiac or skeletal muscle Crush injuries Electric shock Myocardial infarct

47 Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Urine blood/hemoglobin vs myoglobin Both will produce positive reagent strip blood results Marcoscopic differentiation red plasma plus red urine equals hemoglobin clear plasma plus red urine equals myoglobin Chemical differentiation Ammonium sulfate test

48 Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Urine bilirubin formation Formed by hemoglobin degradation bound to albumin and transported through the blood to the liver. unconjugated bilirubin is insoluble in water and cannot be filtered through the glomerulus. conjugated with glucuronic acid in the liver to form bilirubin diglucuronide. Conjugated bilirubin (direct) is water soluble and is excreted by the liver through the bile duct and into the duodenum.

49 Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Urine bilirubin small amounts of conjugated bilirubin regurgitate back from the bile duct and into the blood system. filtered through the glomerulus and excreted in the urine whenever the plasma level is increased. Normally, no detectable amounts are present in urine

50 Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Urine bilirubin Bilirubin color chart acid Bilirubin + diazide azobilirubin

51 Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Urine bilirubin: false positive Technique errors –Reading after the prescribed time atypical color reactions produced by: –Indican –Metabolites of etodolac (Lodine) –Chlorpromazine (Thorazine) –Metabolites of phenazopyridine Confirm results with Ictotest

52 Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Urine bilirubin: false negative Large amounts of ascorbic acid decrease the sensitivity High levels of nitrite Exposure to light and room temperature –Bilrubin oxidizes to biliverdin

53 Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Urine bilirubin confirmatory test Ictotest color reactions.

54 Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Urine bilirubin: significance Normally, no bilirubin is present in urine Present in urine: –when bile flow to colon is obstructed –liver damage –Hepatitis –cholestasis

55 Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Urine urobilinogen formation Formed from bilirubin in the intestines Urobilinogen (colorless) oxidizes to urobilin (brown) Most lost in the feces About 10–15% reabsorbed into the bloodstream, returns to the liver, and reexcreted into the intestines A small amount of this urobilinogen is excreted by the kidneys into the urine Normal level: 1–4 mg/24 h or less than 1.0 Ehrlich unit

56 Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Urine urobilinogen Urobilinogen color chart p-Dimethylaminobenzaldehyde + urobiligen = azo dye

57 Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Urine urobilinogen: false positive p-aminosalicylic acid sulfonamides, p-aminobenzoic Phenazopyridine Prophobilionogen –May use Watson-Shwartz test to differeniate

58 Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Urine urobilinogen: false decrease A true absence of urobilinogen is not detectable. Broadspectrum antibiotics Nitrite Formalin Improper storage of specimen – oxidation of urobilinogen to urobilin

59 Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Urine urobilinogen: significance Normally present in low amounts Peak levels between 2-4 pm Elevated in –Liver disease –Intestinal obstruction –Hemolytic anemia –Hemolysis

60 Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Urine nitrite formation Nitrates normally present Nitrates converted to nitrite by bacteria Reaction takes up to 4 hours to complete UTIs of only non-nitrate reducers will be negative

61 Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Urine nitrite Nitrite color chart Reaction A: Nitrite + p-arsanilic acid diazonium compound Reaction B: 3-Hydroxyl-1,2,3,4 tetrahydrobenz-(h)-quinoline + diazonium compound = pink color

62 Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Urine nitrite: false positive Specimen left at room temperature causing bacteria to multiple. Phenazopyridine

63 Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Urine nitrite: false negative Specimen containing non-nitrate reducing pathogens Insufficient time in the bladder Low or no nitrate diet Nitrite was to nitrogen Elevated urobilinogen Ascorbic Acid

64 Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Urine nitrite: significance Screening method only Not to replace microbiology procedures If there are clinical symptoms, then regular bacteriology tests should be performed, even if the nitrite test is negative.

65 Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Urine leukocyte esterase Leukocyte esterase is present only in neutrophils. Few neutrophils can be seen in normal urine. Increased numbers of neutrophils usually indicate the presence of a urinary tract infection. Screening for UTI also includes pH, protein, and nitrite. Mix specimen well and test at room temperature.

66 Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Urine leukocyte esterase Leuk. Esterase color chart Reaction A: Indoxyl granulocytic indoxyl or carbonic acid ester or pyrole esterase pryole Reaction B: indoxyl or + diazonium salt = purple pyrole

67 Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Urine leukocyte esterase: false positive Strong oxidizing agents Contamination by vaginal discharge Formalin used as preservative Drugs containing imipenem, meropenem, or clavulanic acid Nitrofurantoin contributes a color to urine that may cause misinterpretation

68 Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Urine leukocyte esterase: false negative High specific gravity and in urines containing glucose and protein. –WBCs creanate and cannot release esterase Ascorbic acid Oxalic acid Cephalexin Cephalothin Gentamicin Tetracycline

69 Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Additional urine chemistry parameters Calcium Creatinine Microalbumin Ascorbic acid

70 Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Summary Technological advances have provided for the development of reagent test strips (dipsticks) that allow for the rapid simultaneous determination of commonly measured urinary substances. Abnormal urine chemistry results not only aid in the assessment of renal disorders but can also disclose many systemic disorders. Adhere carefully to all manufacturer instructions to avoid reporting of misinterpreted results.


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