1. UA Chemistry

2. Chemical Analysis Urine Dipstick Glucose Bilirubin Ketones
Specific Gravity
Blood pH
Protein
Urobilinogen
Nitrite
Leukocyte Esterase

3. Improper Technique Errors
RBCs and WBCs sink to the bottom of an unmixed specimen
Enzyme reactions on strip are based on room temperature readings
Reagents will leach off a strip remaining in the urine too long - Dip briefly into specimen
Excess urine on the strip will cause runover of reagents among the pads
The amount of time for reactions to occur is specified by the manufacturer; leukocyte esterase is the longest at 2 minutes

4. Horseradish Peroxidase
The Urine Dipstick:
Glucose
Chemical Principle
Negative
Trace (100 mg/dL)
+ (250 mg/dL)
++ (500 mg/dL)
+++ (1000 mg/dL)
++++ (2000+ mg/dL)
Glucose Oxidase
Glucose + 2 H2O + O2 --->
Gluconic Acid + 2 H2O2
Horseradish Peroxidase
3 H2O2 + KI ---> KIO3 + 3 H2O
Read at 30 seconds
RR: Negative

5. Significance Limitations Other Tests Clinitest
Uses and Limitations of Urine Glucose Detection
Significance
Diabetes mellitus.
Renal glycosuria.
Limitations
False Neg: reducing agents, ketones.
False Pos: oxidizing detergents
Only measures glucose and not other sugars.
Renal threshold must be passed in order for glucose to spill into the urine.
Other Tests Clinitest
CuSO4 test for reducing sugars.

6. The Urine Dipstick: Bilirubin Negative Chemical Principle + (weak)
++ (moderate)
+++ (strong)
Chemical Principle
Acidic
Bilirubin + Diazo salt > Azobilirubin
Read at 30 seconds
RR: Negative

7. Acetoacetic Acid + Nitroprusside ------> Colored Complex
The Urine Dipstick:
Ketones
Negative
Trace (5 mg/dL)
+ (15 mg/dL)
++ (40 mg/dL)
+++ (80 mg/dL)
++++ (160+ mg/dL)
Chemical Principle
Acetoacetic Acid + Nitroprusside
------> Colored Complex
Read at 40 seconds
RR: Negative

8. The Urine Dipstick: Specific Gravity 1.000 Chemical Principle 1.005
1.010
1.015
1.020
1.025
1.030
Chemical Principle
X+ + Polymethyl vinyl ether / maleic anhydride
>
X+-Polymethyl vinyl ether / maleic anhydride + H+
H+ interacts with a Bromthymol Blue indicator to
form a colored complex.
Read up to 2 minutes
RR:

9. Uses and Limitations of Urine Specific Gravity
Significance
- Diabetes insipidus
Limitations
- Interference: alkaline urine
- Does not measure non-ionized solutes (e.g. glucose)
Other Tests
- Refractometry
- Hydrometer
- Osmolality measurement (typically used with water deprivation test)

10. The Urine Dipstick: Blood Negative Trace (non-hemolyzed)
Chemical Principle
Trace (non-hemolyzed)
Lysing agent to lyse red blood cells
Moderate (non-hemolyzed)
Diisopropylbenzene dihydroperoxide +
Tetramethylbenzidine
> Colored Complex
Trace (hemolyzed)
Heme
+ (weak)
++ (moderate)
Read at 60 seconds
RR: Negative
Analytic Sensitivity: 10 RBCs
+++ (strong)

11. Uses and Limitations of Urine Blood Detection
Significance
- Hematuria (nephritis, trauma, etc)
- Hemoglobinuria (hemolysis, etc)
- Myoglobinuria (rhabdomyolysis, etc)
Limitations
- Interference: reducing agents, microbial peroxidases
- Cannot distinguish between the above disease processes
Other Tests
- Urine microscopic examination
- Urine cytology

12. Methyl Red (at high concentration; low pH) and
The Urine Dipstick: pH
H+ interacts with:
Methyl Red (at high concentration; low pH) and
Bromthymol Blue (at low concentration; high pH), to form a colored complexes (dual indicator system)
Chemical Principle
Read up to 2 minutes
R.R.:
5.0
6.0
6.5
7.0
7.5
8.0
8.5

13. Uses and Limitations of Urine pH Detection
Significance
- Acidic (less than 4.5): metabolic acidosis, high-protein diet
- Alkaline (greater than 8.0): renal tubular acidosis (>5.5)
Limitations
- Interference: bacterial overgrowth (alkaline or acidic),
“run over effect” effect of protein pad on pH indicator pad
Other Tests
- Titrable acidity
- Blood gases to determine acid-base status

14. pH Run Over Effect Glucose Bilirubin Ketones
Specific Gravity
Blood pH
Protein
Urobilinogen
Nitrite
Leukocyte Esterase
Buffers from the protein area of the strip (pH 3.0) spill over to the pH area of the strip and make the pH of the sample appear more acidic than it really is.

15. The Urine Dipstick: Protein Chemical Principle
“Protein Error of Indicators Method”
Negative
Trace
+ (30 mg/dL)
++ (100 mg/dL)
+++ (300 mg/dL)
++++ (2000 mg/dL) Pr H
Tetrabromphenol Blue
(buffered to pH 3.0) H+ Pr Pr Pr Pr Pr Pr
Read at 60 seconds
RR: Negative

16. Uses and Limitations of Urine Protein Detection
Significance
- Proteinuria and the nephrotic syndrome.
Limitations
- Interference: highly alkaline urine.
- Much more sensitive to albumin than other proteins
(e.g., immunoglobulin light chains).
Other Tests
- Sulfosalicylic acid (SSA) turbidity test.
- Urine protein electrophoresis (UPEP)
- Bence Jones protein

17. Urobilinogen + Diethylaminobenzaldehyde -------> Colored Complex
The Urine Dipstick:
Urobilinogen
0.2 mg/dL
1 mg/dL
2 mg/dL
4 mg/dL
8 mg/dL
Chemical Principle
Urobilinogen + Diethylaminobenzaldehyde
> Colored Complex
(Ehrlich’s Reagent)
Read at 60 seconds
RR: mg/dL

18. Uses and Limitations of Urobilinogen Detection
Significance
- High: increased hepatic processing of bilirubin
- Low: bile obstruction
Limitations
- Interference: prolonged exposure of specimen to oxygen (urobilinogen ---> urobilin)
- Cannot detect low levels of urobilinogen
Other Tests
- Serum total and direct bilirubin

19. The Urine Dipstick: Nitrite Chemical Principle Negative Positive
Acidic
Negative
Positive
Nitrite + p-arsenilic acid > Diazo compound
Diazo compound + Tetrahydrobenzoquinolinol
> Colored Complex
Read at 60 seconds
RR: Negative

20. Uses and Limitations of Nitrite Detection
Significance
- Gram negative bacteriuria
Limitations
- Interference: bacterial overgrowth
- Only able to detect bacteria that reduce nitrate to nitrite
Other Tests
- Correlate with leukocyte esterase and
- Urine microscopic examination (bacteria)
- Urine culture

21. The Urine Dipstick: Leukocyte Esterase Chemical Principle
Derivatized pyrrole amino acid ester
> 3-hydroxy-5-phenyl pyrrole
Negative
Trace
+ (weak)
++ (moderate)
+++ (strong)
Esterases
3-hydroxy-5-phenyl pyrrole + diazo salt
> Colored Complex
Read at 2 minutes
RR: Negative
Analytic Sensitivity: 3-5 WBCs

22. Significance Limitations Other Tests
Uses and Limitations of Leukocyte Esterase Detection
Significance
- Pyuria
- Acute inflammation
- Renal calculus
Limitations
- Interference: oxidizing agents, menstrual contamination
Other Tests
- Urine microscopic examination (WBCs and bacteria)
- Urine culture

23. Protein
Protein Error of Indicators pH 3 False Positive Highly Buffered Alkaline urine Detergent (Ammonium compounds) Loss of Buffer
False Negative Proteins other than Albumin

24. Protein
Sulfosalicylic Acid Precipitation False Pos Radiographic Dye Sp Gr
Bence Jones Heat Prec then redisolve at 100
Electrophoresis
Microalbuminuria Diabetic Screen

25. Protein Clinical Significance
Prerenal Hemolysis, Muscle Injury, Multiple Myeloma, Infection
Renal Glomerular Tubular
Postrenal UTI, Injury, Contamination

26. Protein Clinical Significance
Protein is present in just about all renal diseases.
Expect a positive protein if Casts are in sediment.
Orthostatic Proteinuria