1. Clinical Urinalysis Review
Austin Community College
Medical Laboratory Technology
Clinical II Spring 07

2. Urine Blood Testing

3. Chemical Exam of Urine

4. Chemical Exam of Urine

5. Chemical Exam of Urine Reagent strip manufactures
Bayer Corporation- Diagnostics Division (formerly Ames) produces Multistix
Boehringer-Mannheim Corporation which produces Chemstrip
Behring Diagnostics which produces Rapignost

6. Chemical Exam of Urine

7. Chemical Exam of Urine Reagent strip precautions and source of errors
Normal dipstick procedure:
Dip strip briefly, but completely into well mixed, room temperature urine sample.
Withdraw strip, blot briefly on its side
Keeping the strip flat, read results at the appropriate times by comparing the color to the appropriate color on the chart provided.

8. Chemical Exam of Urine Sources of error (& preventions)
Testing cold specimens
would result in a slowing down of reactions; test specimens when fresh or bring them to RT before testing
Inadequate mixing of specimen
could result in false reduced or negative reactions to blood and leukocyte tests; mix specimens well before dipping
Over-dipping of reagent strip
will result in leaching of reagents out of pads; briefly, but completely dip the reagent strip into the urine

9. Chemical Exam of Urine Improper timing of tests
Inadequate blotting & Failure to keep strip horizontal
will result in over-run or mixing of reagents between the different reaction pads; blot excess urine off the strip and keep strip horizontal. If dipping from the tube, can run the side of the strip along the rim to remove excess urine.
Improper timing of tests
over development of reagent pad colors leading to falsely increased results; follow manufacturer’s recommendations

10. Chemical Exam of Urine Inadequate light Mis-using color chart
misinterpretation of results; use good lighting
Mis-using color chart
misinterpretation of results; hold strip just over color chart and match colors as close as possible, consider use of back-up tests, if needed, especially if urine’s color masks reaction colors.

11. Chemical Exam of Urine Handling and Storage
Keep strips in original container, stored at RT
Protect from moisture and volatile fumes
Use before expiration date
Do not touch reagent pad areas

12. Chemical Exam of Urine
Quality Control - use appropriate, commercially prepared positive and negative controls.
Use commercially prepared pos and neg controls, at least once per 24 hours, and anytime a new bottle is opened, or question of validity of results. Readings should agree with published results ± one color block.

13. Urine Glucose Testing Normal : no glucose detected
Clinical significance of abnormal results (Glucosuria)
Plasma glucose level exceeds renal threshold ( mg/dL)
Diabetes mellitus
Renal tubular dysfunction
Filtered glucose not being reabsorbed in tubules

14. Urine Glucose Testing Dipstick Testing Method
Glucose initiates reaction
Coupled reaction
Glucose oxidase – oxidizes glucose to gluconic acid and concurrently reduces oxygen to hydrogen peroxide.
Hydrogen peroxide in presence of the enzyme peroxidase will oxidize an indicator, giving a colored reaction.
Chromogens
Potassium iodide or
Tetramethylbenzindine

15. Urine Glucose Testing Sensitivity
@ mg (compared to Clinitest’s 250) SO- Can have a positive dipstick but a neg Clinitest
Specificity - is specific for glucose only.
not affected by other sugars or reducing substances.

16. Urine Glucose Testing Interfering substances
High specific gravity and high pH may depress color.
Ascorbic acid-false neg
Bleach or peroxide may give false positive

17. Urine Bilirubin Testing
Normal : no bilirubin detected
Clinical significance of abnormal results (Bilirubinuria)
Jaundice - Condition when serum bilirubin becomes greater than the liver can handle, and there is an abnormal collection of bilirubin in the tissues giving them a yellow color

18. Urine Bilirubin Testing
Prehepatic / Hemolytic jaundice
Excessive hemolysis of RBC; beyond what the liver can process
Type of bilirubin?
Is bilirubin found in the urine? YES/NO? Explain.

19. Urine Bilirubin Testing
Prehepatic / Hemolytic jaundice
Type of bilirubin? – indirect, insoluble, unconjugated
Is bilirubin found in the urine? – No, the bilirubin is not water soluble

20. Urine Bilirubin Testing
Hepatic jaundice
Liver’s cells malfunctioning
Ie. viral hepatitis, cirrhosis etc.
Both (direct) bilirubin and urobilinogen found in urine.

21. Urine Bilirubin Testing
Post hepatic (regurgative or obstructive) hepatitis
Obstruction to outflow of bile – some type of blockage
Gall stones
Tumor
Edema
Conjugated bilirubin backed up into blood (Bilirubinuria) and passes into urine

22. Urine Bilirubin Testing
Testing method
Urine dipsticks for bilirubin – a diazo reaction
Impregnated with stabilized diazotized 2,4 dichloraniline
Color goes from buff to brown also shades of pink – violet
If urine is strongly colored, look for change in pad color after dipping. Use Ictotest for backup.

23. Urine Bilirubin Testing
Interfering substances
Medication metabolites, pigments and indican may obscure readings
False negatives due to aged specimens, especially those exposed to light and oxidation.

24. Urine Ketone Testing Ketone Bodies
Origin - not normally present
Products of fat catabolism - breakdown of fat into CO2 and H2O
What are the 3 ketone bodies?

25. Urine Ketone Testing Acetone Diacetic Acid (Acetoacetic)
2%. -Acetone is volatile, & excreted primarily through the lungs
Diacetic Acid (Acetoacetic)
the first formed,
20 % of the total
the form detected by most ketone test procedures
Beta hydroxybutyric Acid
majority formed, but not detected by routine tests.
Only Hart’s test, an old ‘wet chemical’ test will detect this one.

26. Urine Ketone Testing Definitions Ketonuria - ketones in the urine
Ketonemia - ketones in the blood
Ketosis - disease state, when patient has increased amount of ketones.
Acidosis - state when blood pH is decreased, an accumulation of acids; commonly occurs as a result of ketosis

27. Urine Ketone Testing Clinical significance High fat diet
Health – formed in liver and completely metabolized
Disease – excessive formation and accumulation
Disturbance of carbohydrate metabolism
when there is a decrease of carbohydrate metabolism, then the body stores of fat must be metabolized to supply energy.
As a result of this increased fat metabolism ketones will be found in the urine. Ex. low carbohydrate diets, diabetes
Starvation
Vomiting and diarrhea in children
Van Gierke's Disease – glycogen storage disease
High fat diet

28. Urine Ketone Testing Clinical effects Metabolic acidosis
Lowering of blood & urine pH
Brain toxicity

29. Urine Ketone Testing Testing most use nitroprusside
detects diacetic acid and a small amount of acetone, but does not detect β-hydroxybutyric acid.
Produces purple color
Can be used on urine or blood

30. Urine Specific Gravity Testing
The specific gravity is a measure of the weight of urine compared to an equal amount of water. 
Specific gravity it proportional to urine osmolality which is a measure of concentration.

31. Urine Specific Gravity Testing
The specific gravity will always be greater than and will increase as more materials are dissolved in the urine. 
The value changes throughout the day depending on fluid intake.

32. Urine Specific Gravity Testing
Specific gravity between & on a random sample is normal if kidney function is normal.
Specific gravity in Bowman’s capsule fluid 1.007
Any reading below this indicates hydration
Any reading above this indicates some degree of dehydration

33. Urine Specific Gravity Testing
Again dilute urine will have values less than  
Fixed specific gravity = 1.010; isothenuria
Diabetes insipidus
End-stage renal disease
And concentrated urine will have values usually over
Usually due to dehydration and can be seen in well population as well as sick.

34. Urine Specific Gravity Testing
Increased urine specific gravity may indicate / be seen in:
* Dehydration
* Diarrhea
* Excessive sweating
* Glucosuria
* Heart failure (related to decreased blood flow to the kidneys)
* Renal arterial stenosis
* Syndrome of inappropriate antidiuretic hormone secretion (SIADH)
* Vomiting
* Water restriction

35. Urine Specific Gravity Testing
Decreased urine specific gravity may indicate / be seen in:
* Excessive fluid intake
* Diabetes insipidus – central or nephrogenic
* Renal failure (that is, loss of ability to reabsorb water)
* Pyelonephritis

36. Urine Specific Gravity Testing
Specific gravity > (refractometer)
Could have very high glucose levels
Could contain radiographic dye

37. Urine Specific Gravity Testing
Polyelectrolytes , pH indicator (bromthymol blue measures the pH change), and alkaline buffer

38. Urine Specific Gravity Testing
Interfering substances
False elevation of results may be seen in samples with increased protein concentration.
Some reports of reduced specific gravity results on alkaline specimens.
Lipids may also effect results

39. Urine Blood Testing Normally not found in urine
Hemoglobinuria – free hemoglobin in urine
Circulating free hemoglobin normally picked up by haptoglobin preventing loss in urine
When serum levels of hemoglobin > 100 mg/dL threshold is exceeded
Hematuria – RBCs in the urine
Trauma / irritation of renal organs

40. Urine Blood Testing

41. Urine Blood Testing
Testing dipstick reaction

42. Urine Blood Testing ‘Blood’ test detects
Free Hemoglobin
RBCs – get lysed on the pad & their hemoglobin reacts
Myoglobin – muscle hemoglobin
Principle based on the peroxidase-like activity of the heme portion of the molecule

43. Urine Blood Testing
Sensitivity – can detect at levels of 5-10 cells/uL
Interfering substances
Ascorbic acid
Nitrates
Oxidizing agents (ie bleach)
Contaminate blood (menstrual)

44. Urine pH Testing Normal: kidneys capable of 4.5 – 8.0
Factors effecting pH
Diet – general & specific foods
Time of day
Metabolic disorders
Drugs / medications
Dipstick capable: 4.5 – 9.0

45. Urine pH Testing Test method
Dipstick indicators – methyl red and bromthymol blue
Range
Caution – other chemicals on dipstick can effect pH reading

46. Urine Protein Testing
Normally not found in measurable amounts on dipstick (<150 mg/dL /day)
Permeability of glomerulus
Damage to glom capularies
Changes in glom blood flow
Albumin excretions may be increased temporarily due to exercise, uti, and acute illness with fever.
Dipstick results of 1+ (30mg/dL) would equal 500 mg/dL (clinical proteinuria)

47. Urine Protein Testing Only albumin detectable by dipstick
Sensitivity ml/dL)

48. Urine Protein Testing New testing for microalbumin & creatinine
Results:
Protein mg/dL ( mg/dL /24 hr)
Creatinine mg/dL
Albumin/creatinine ratio
Normally albumin in the urine is less than 30 mg/ gram creatinine

49. Urine Protein Testing Principle - Protein error of indicators
at fixed pH, certain indicators show one color in the presence of protein and another in absence of protein - the “error” of the indicator.
Indicator – tetrabromphenol blue - can be hard to read at the trace end
Citrate Buffer – maintains pH 3 -quite acid

50. Urine Protein Testing Sources of error Sensitive only to albumin
Urine with strong / unusual color makes reading difficult
Highly alkaline or buffered urine will neutralize acid buffer and lead to increased erroneous results.
Urine container contamination would interfere

51. Urine Protein Testing Urine back up test 3% sulfosalicylic acid
Added to the supernatant to detect any kind of protein. Urine will turn cloudy if protein is present.

52. Urine Urobilinogen Testing
Normally found in small amounts, especially in early afternoon
Increased amounts may indicate liver disease or be seen as result of hemolytic disorders
Decreased amounts:
If intestinal bacteria destroyed
Liver doesn’t conjugate bilirubin
Biliary obstruction – failure of bilirubin to reach small intestine

53. Urine Urobilinogen Testing
Test principle based on Ehrilich’s reaction
Para-dimethylaminobenzaldehyde = Ehrlich's reagent.
Must protect specimen from light and test immediately

54. Urine Nitrate Testing Nitrate
Detects presence of certain types of bacteria
screening for presence of UTI.
Certain species of bacteria convert nitrate (normal constituent of urine) to nitrite
Escherichia - most common cause of UTI
Klebsiella
Proteus
Pseudomonas
Enterobacter
Citrobacter

55. Urine Nitrate Testing
Aromatic amine in reagent strip reacts with nitrite; producing a diazonium salt
The diazonium salt reacts with sulfanilic acid and acetic acid to produce a pink azo dye

56. Urine Nitrate Testing Limitations reported as positive or negative
Not all UTI causing bacteria convert nitrate to nitrite
Haemophilus
Staphylococcus
Streptococcus

57. Urine Nitrate Testing
Fresh first morning specimen is preferred - besides being the most concentrated specimen, the urine has been in the bladder longer, allowing bacteria time and opportunity to convert the nitrates to nitrites.

58. Urine Leukocyte Testing
Leukocyte esterase testing is another test used as a means of screening for urinary tract infection.
Does not measure concentration of leukocytes
Will detect presence of lysed leukocytes as well as intact WBCs

59. Urine Leukocyte Testing
test principle:
Leukocyte esterase, an enzyme present in granulocytes, hydrolyzes indoxylcarbonic acid esterase to produce indoxyl, which reacts with a diazonium salt to create a purple color usually in 2 min.

60. Urine Leukocyte Testing
Reaction interference
False positives - oxidizing detergents
False negatives - greatly increased glucose, protein, or specific gravity- increased sp gr could cause WBC to crenate preventing their releasing their esterase, So it is possible for the dipstick to be negative when there are WBCs present.