1. Chemical Examination of Urine
Ricki Otten MT(ASCP)SC
CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

2. Objectives: Review the objectives on page 1 and 2
of the lecture handout
Objectives marked with ‘*’ will not be tested over during student lab rotation
CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

3. Historical Perspective: Urinalysis
Physical examination of urine
Odor
Taste
Color
Clarity
CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

4. Historical Perspective
Chemical examination of urine
Limited reactions
Required large volumes of urine
Large volumes of reagent
Performed in test tubes
Time consuming and cumbersome
Clinical usefulness was not realized
Not routinely ordered
CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

5. Historical Perspective
Microscopic examination of urine
Not until invention of the microscope
Then clinical usefulness realized
CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

6. Reagent Strip Testing Technology and necessity
Chemical reactions ‘miniaturized’
Required less urine
Test results within minutes
Easy to perform
Increased test utilization
Brunzel, 2nd Ed, page 124
CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

7. Reagent Strip Testing Ideal qualitative screening tool
Sensitive: Low concentration of substances
Negative result = normal
Specific: Reacts with only one substance
False negative and false positive
Cost effective: Relatively inexpensive tool that
provides information about the health status
of the patient
CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

8. Reagent Strip Testing Chemically impregnated absorbent
pads attached to an inert plastic strip
Each pad is a specific chemical reaction that
takes place upon contact with urine
Chemical reaction causes the color of the pad to
change
Color compared to a color chart for interpretation
CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

9. Reagent Strip Testing Qualitative or semi-quantitative results
Concentration units (mg/dl)
Negative, small, moderate large
Negative, 1+, 2+, 3+, 4+
Timing of chemical reactions is CRITICAL
Shortest time requirement on one end of strip: 30 sec
Longest time requirement on the other: 2 min
CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

10. Reagent Strip Testing Principle of chemical reactions
False negative reactions
False positive reactions
Color interferences
Alternative testing: used to confirm results that you may think are invalid due to
Interfering substance
Color interference (called color masking)
CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

11. Confirmatory Testing (pg 6)
Care and Storage (pg 4)
Reading assignment:
Textbook, chapter 7
Page
Confirmatory Testing (pg 6)
CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

12. Confirmatory Testing
Alternative testing establishes the correctness or accuracy of another procedure
Often used when urine is highly pigmented
Bilirubin reagent strip  ictotest
CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

13. Confirmatory Testing Ideallywant all 3 Characteristics:
Differ in sensitivity
Ictotest vs Bilirubin reagent strip
Differ in specificity
SSA vs Protein reagent strip
Clinitest vs Glucose reagent strip
Differ in methodology/reaction
Ideallywant all 3
CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

14. Differ in Specificity Clinitest reacts with all reducing substances
Glucose reagent strip reacts with only one reducing substance: glucose
CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

15. Causes of invalid results
10 reagent strip tests
Specific gravity pH
Protein
Glucose
Ketones
Blood
Bilirubin
Urobilinogen
Nitrite
Leukocyte Esterase
Purpose of the test
What is normal
What is abnormal
Reaction
Causes of invalid results
CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

16. Specific Gravity: Purpose
Evaluates the concentrating and diluting
ability of the kidney
Density is related to the amount of substances (solutes) in solution
Increased density ~ increased solute in solution ~ hypertonic urine ~ concentrated urine
Decreased density ~ decreased solute in solution ~ hypotonic urine ~ dilute urine
CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

17. Specific Gravity: Normal
Majority of urines: – 1.025
Physiologically impossible: 1.000
>1.040
Dependent upon hydration status
CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

18. Specific Gravity: Terms
Isosthenuria
Fixed at 1.010
Renal tubules lost absorption and secreting capability
Hypersthenuria
Increased specific gravity
Concentrated urine
Hyposthenuria
Decreased specific gravity
Dilute urine
Sensitivity issues:
Pregnancy testing
Urinary tract infection
CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

19. Specific Gravity: Methods
Methods of measurement
Reagent strip test: indicates ionic solutes
Refractometer: indicates amount of total solutes
Two functions of the kidney
Maintain water balance
Maintain electrolyte homeostasis
Performed by renal tubules through concentrating and diluting; reabsorbing and secreting water and electrolytes (ionic)
CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

20. Specific Gravity: Reaction
Based on a change in the pKa of a polyelectrolyte on the reagent pad
Increased ions in solution causes the polyelectrolyte on the pad to produce free H+
Free H+ cause a change in pH on the reagent pad
Change in pH: bromthymol blue indicator
CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

21. Specific Gravity: Reaction
CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

22. Specific Gravity Sensitivity: 1.000
Specificity: detects only ionic substances
Radiographic dye
Mannitol
Glucose
Does not interfere
CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

23. pH: Purpose Kidneys regulate body’s acid-base
balance by selective handling of H+ and HCO3-
Urine pH reflects acid-base status of body
Treatment protocol may require urine pH be maintained at a specific pH
(Aids in identification of crystals (microscope))
CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

24. pH: Normal Normal: ranges from 4.5 – 8.0 First morning void: acidic
Physiologically impossible: <4.5
>8.0
Urine not handled properly
Old urine
Treatment induced
CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

25. pH: Interpretation Made in conjunction with Acid-base status
Renal function
Presence of infection in urinary tract
Diet: high protein, low protein
Medications
Age of urine sample
CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

26. pH: Abnormal Acid Alkaline Respiratory acidosis High protein diet
Starvation
UTI
Alkaline
Respiratory alkalosis
Vegetarian diet
Renal tubular acidosis
UTI
CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

27. pH: Reaction Double indicator system
Methyl red
Bromthymol blue
Amount of free H+ influences acidity of urine and cause pH indicator to change color
Needed to measure the wide pH range: acid to alkaline
CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

28. pH: Invalid test results due to: Improper handling of urine sample
Contamination of urine vessel prior to collection
‘Run-over’ phenomenon
CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

29. Protein: Purpose Normal kidneys secrete LITTLE protein
<15 mg/dl (or <150 mg/24 hours)
The protein that is found in urine comes from
Bloodstream
Urinary tract
Proteinuria is an indicator of early renal disease
Proteinuria also caused by non-renal disease
CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

30. Renal Cause of Proteinuria:
Glomerular damage:
Most serious cause of proteinuria
Most common cause of proteinuria
Glomerulonephritis
Nephrotic Syndrome
Tubular dysfunction:
Reabsorption capability decreased
Toxin exposure, inherited disorder
Fancon’s syndrome: heavy metal poisoning
CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

31. Classification of Proteinuria
Functional
Orthostatic (postural)
Transient
Pathologic
Pre-renal (overflow)
Renal: glomerular
Renal: tubular
Post-renal
CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

32. Protein: Methods Reagent strip test SSA test Foam test
Micro-albumin test
CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

33. Protein: Reagent Strip
The reagent pad is held at a
constant pH of 3 by a buffer
Proteins (anions) in solution cause an
indicator dye to release H+ causing a color
change
‘Protein error of indicators’
CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

34. Protein: Reagent Strip
Sensitivity: ~ mg/dl
Specificity: reacts with albumin
False positive: highly alkaline urine (pH > 8.0)
False negative:
Dilute urine
Presence of other proteins
(Tamm-Horsfall, globulins, myoglobin,
free light chains, hemoglobin)
CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

35. Protein: SSA (Exton’s Test)
Sulfosalicylic Acid (SSA) Precipitation Test
Acid will precipitate proteins out of solution causing the solution to become cloudy
Amount of cloudiness is related to the amount of protein present
CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

36. Protein: SSA (Exton’s Test)
Amount of cloudiness is evaluated, thus must use centrifuged urine
Sensitivity: 5-10 mg/dl
Specificity: detects all protein
CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

37. Protein: SSA (Exton’s Test)
False positive results:
Radiographic dyes
Turbid urine
Uncentrifuged urine
False negative results:
Highly alkaline urine
Dilute urine
CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

38. Protein: Foam test
Shake aliquot of urine and observe color of resulting foam
White foam: protein present
CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

39. Protein: Micro-albumin test
Measures very low concentration of albumin (better sensitivity than reagent strip test for albumin)
Management of diabetic patient
Methods vary: reagent strip test,
immunochemical reaction
CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

40. Glucose: Purpose Healthy normal urine does not contain glucose
Normally, glucose is filtered by the glomerulus and is reabsorbed back into the bloodstream through active transport mechanism
Glucose in urine is pathologic
CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

41. Glucose: Purpose Glucosuria Glycosuria
Caused by renal and non-renal disease
Pre-renal glycosuria: plasma glucose level exceeds renal threshold (diabetes mellitus)
Renal glycosuria: plasma glucose level below renal threshold, but tubules cannot reabsorb glucose back into bloodstream
Terms used interchangeably
CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

42. Reducing Substances: Purpose
Glucose
Other sugars: galactosemia (inherited metabolic disorder)
CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

43. Glucose, Reducing Substances
Normal: negative
Abnormal:
Diabetes mellitus: glucose
Impaired renal tubular reabsorption: glucose
Inborn error of metabolism: galactosemia
CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

44. Methods Reagent strip: detects only glucose
Copper Reduction: detects reducing substances
CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

45. Glucose: Reagent Strip
Detects only glucose
Double sequential enzyme reaction
CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

46. Glucose: Reagent Strip
Sensitivity: ~ 30 mg/dl
Specificity:
Reacts only with glucose
False positive:
Strong oxidizing agents (bleach)
Peroxides
False negative:
Ascorbic acid (reducing agent)
Improperly stored urine: glycolysis
CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

47. Clinitest Reaction Copper Reduction Test:
Reducing substances are able to reduce copper sulfate to cuprous oxide
Pass-through phenomenon
All children <2 years: metabolic disorder
(galactosemia)
CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

48. Clinitest Reaction Sensitivity: ~ 250 mg/dl Specificity:
Reacts with all reducing substances
Reducing sugars: glucose, galactose, fructose, lactose, maltose (NOT SUCROSE)
False positive: any reducing substance
(Ascorbic acid)
False negative: radiographic dye
CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

49. Ketones: Purpose Ketones are intermediary products of fat metabolism
CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

50. Ketones Three ketone bodies Characteristic ‘fruity breath’ ~ acetone
Acetoacetic acid 20%
Beta-hydroxybutyric acid 78%
Characteristic ‘fruity breath’ ~ acetone
CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

51. Ketones: Normal Normal: negative Abnormal:
Inability to utilize carbohydrates
Excessive loss of carbohydrates
Inadequate intake of carbohydrates
CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

52. Ketones: Methods Reagent strip Acetest: tablet test
CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

53. Ketones: Method Glycine: also measures acetone
Reagent strip: check package insert
Acetest tablets: contain glycine
CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

54. Ketones Reagent strip Acetest Sensitivity: 5-10 mg/dl
Specificity: acetoacetic acid and/or acetone
False positive: highly pigmented urine
False negative: improper specimen handling
Acetest
Specificity: acetoacetic acid and acetone
False positive: highly pigmented urien
CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

55. Blood: Purpose Blood in urine indicates pathology
Two forms found in urine
Intact RBC
Hemolyzed RBC
CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

56. Blood: Terms Hematuria Hemoglobinuria Myoglobinuria
All will give a positive blood reaction
CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

57. Blood: Reagent strip Test can detect hemolyzed RBC
Heme moiety imparts peroxidase activity and catalyzes the reaction
CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

58. Blood Sensitivity Specificity Intact RBC Hemolyzed RBC (hemoglobin)
Myoglobin
False positives: myoglobin, oxidizing agents
False negatives: ascorbic acid
CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

59. Blood: Correlate reagent strip results Microscopic findings
Color and clarity
CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

60. Bilirubin and Urobilinogen
Bilirubin in urine is always pathologic:
liver disease
Urobilinogen in urine: normal to have a small amount:
0.2 – 1.0 mg/dl
CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

61. Three mechanisms Pre-hepatic: liver is healthy Hepatic: liver disease
Post-hepatic: liver is healthy, obstruction indicated
CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

62. Bilirubin: Methods Reagent strip Ictotest: tablet test Foam test
CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

63. Bilirubin: Methods Reagent strip Ictotest: tablet test Same reaction
Same specificity: conjugated bilirubin
False positive: urine color
False negative: low concentration, ascorbic acid, improper specimen handling
CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

64. Bilirubin: Methods Reagent strip Ictotest: tablet test
Sensitivity differs
Reagent strip: ~0.5 mg/dl
Ictotest: – 0.1 mg/dl
CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

65. Bilirubin: Methods
Possible to have a negative reagent strip test and positive ictotest
Difference in sensitivity levels
Always perform Ictotest when
Urine bilirubin test specifically ordered
Urine appearance is amber: even if bilirubin
reagent strip test is negative
Positive reagent strip test
CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

66. Bilirubin: Foam Test Shake urine and observe resulting foam
Yellow foam = bilirubin
CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

67. Urobilinogen: Methods
Reagent strip test
Two reactions dependent upon manufacturer
Para-dimethylaminobenzaldehyde
Diazonium salt
Cannot determine absence of UBG
Watson-Schwartz assay
CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

68. Urobilinogen: Methods
Para-dimethylaminobenzaldehyde
Sensitivity: 0.2 mg/dl
Specificity:
False positive: any ‘Ehrlich reactive compound’; color masking; urine at body temp
False negative: improper specimen handling
Diazonium salt
Sensitivity: 0.4 mg/dl
Specificity: reacts only with UBG
False positive: color masking
CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

69. Urobilinogen: Watson Schwartz
Classic method used to differentiate
urobilinogen from porphobilinogen using a
differential extraction method
Para-dimethylaminobenzaldehyde
CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

70. Nitrite: Purpose
Bacteria that contain a specific enzyme can reduce dietary nitrates to nitrites
Rapid screening test for UTI
CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

71. Nitrite: Normal Normal: negative Abnormal: Cystitis: bladder
Pyelonephritis: kidney
CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

72. Nitrite: Method Reagent strip test
Nitrite + aromatic amine  diazonium salt
Diazonium salt + aromatic compound 
pink color
Sensitivity: mg/dl nitrite
~ 10,000 organisms
CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

73. Nitrite: Method Reagent strip test Specificity:
False positive: improper specimen handling; color masking
False negative: bacteria cannot reduce nitrates
Bladder time not sufficient: need 4 hours
Low nitrite levels
Ascorbic acid
Antibiotic inhibition of bacteria
Further reduction of nitrites to nitrogen
CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

74. Leukocyte Esterase: Purpose
Increased WBC in urine is pathologic
Indicates inflammation, infection
Neutrophils most common type of WBC found in urine
Can detect intact WBC and lysed WBC
CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

75. Leukocyte Esterase: Normal
Normal: negative
Abnormal:
Bacterial infection:
cystitis, pyelonephritis, urethritis
Non-bacterial infection: yeast, trichomonas
CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

76. Leukocyte Esterase: Method
Reagent strip:
Granules in cytoplasm of WBC contain an enzyme (esterase)
Ester –esterase aromatic compound
Aromatic compound + diazonium salt 
Purple colored complex
CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

77. Leukocyte Esterase Sensitivity: 5-15 WBC/hpf Specificity:
False positive: vaginal contamination; color masking
False negative: strong oxidizing agents (bleach); lymphocytes (no granules)
CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

78. CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine