1. Ricki Otten MT(ASCP)SC uotten@unmc.edu
Chemical Examination of Urine Part I: Introduction, Specific Gravity and pH
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