Chemical Determination Of Urine Flashcards
Glucose
Reading Time: Principle: Positive Color
30 seconds: Double sequential enzyme reaction: Green to Brown
Bilirubin
Reading Time: Principle: Positive Color
30 seconds: Diazo reaction: Tan or pink to violet
Ketones
Reading Time: Principle: Positive Color
40 seconds: Sodium nitroprusside reaction (Legal’s Test): Purple
Specific Gravity
Reading Time: Principle: Positive Color
45 seconds: PKa change of a polyelectrolyte: Blue (SG 1.000) to yellow (SG 1.030)
Protein
Reading Time: Principle: Positive Color
60 seconds: Protein (Sorensen’s) error of indicators: Blue
pH
Reading Time: Principle: Positive Color
60 seconds: Double indicator system: Orange (pH 5.0) to Blue (pH 9.0)
Blood
Reading Time: Principle: Positive Color
60 seconds: Pseudoperoxidase activity of hemoglobin
Uniform green/blue (Hgb or Mb) Speckled/spotted (intact RBCs)
Urobilinogen
Reading Time: Principle: Positive Color
60 seconds: Ehrlich reaction
Red
Nitrite
Reading Time: Principle: Positive Color
60 seconds: Greiss reaction
Uniform Pink
Leukocytes
Reading Time: Principle: Positive Color
120 seconds: Leukocyte esterase
Purple
Reagent Strip Technique (4)
- Dip the reagent strip in a well-mixed urine (uncentrifuged) (Room temperature) not more than 1 second
- Blot the edge of the reagent strip on disposable absorbent pad
- Wait for each reading time to be reach
- Compare the color per parameter.
Care for Reagent Strips
Store in?
Store below?
Do not?
opaque , tightly closed container
30°C (RT)
freeze
Automated Reagent Strip
Readers principle
Light reflection from the test pads decreases in proportion to intensity of color produced by
the concentration of the test substance
Specific Gravity is Influenced by the
number and size of particles in solution
Normal SG (random)
1.003-1.035 (random)
When SG <1.003
Not a urine (except in D.I)
When SG >1.040
Radiographic dye present
(improve visibility of internal structures for X-ray)
SG = 1.010 is referred to as
Isosthenuria
SG < 1.010 (diluted) is referred to as
Hyposthenuria
SG > 1.010 (concentrated) is referred to as
Hypersthenuria
Urinometry (Urinometer/Hydrometer)
Reading:
Calibration Temperature:
Requires temperature correction:
- Below 3°C=
- Above 3°C=
Requires correction for Glucose and protein:
- 1 g/dL Glucose=
- 1 g/dL Protein=
Urine volume required:
Calibration:
Reading: LOWER MENISCUS Calibration Temperature: 20 °C Requires temperature correction:
❑ Below 3°C= =-0.001
❑ Above 3°C= +0.001
Requires correction for Glucose and protein:
❑ 1 g/dL Glucose= -0.004 (subtract)
❑ 1 g/dL Protein= -0.003 (subtract) Urine volume required: 10- 15 mL
Calibration:
❑ Potassium sulfate (K2SO4) solution= add 20.29g K2SO4 to 1 L of Water=SG should always be 1.015
Indirect method based on refractive index
Refractometry (Refractometer/TS Meter)
Do Refractometry (Refractometer/TS Meter) requires temperature correction?
No
Refractometry (Refractometer/TS Meter) requires correction for?
Glucose and Protein
Refractometry (Refractometer/TS Meter)
Calibration:
Distilled water: SG=
3% NaCl:
5% NaCl:
9% Sucrose:
Calibration:
Distilled water: SG= 1.000
3% NaCl: 1.015 ± 0.001
5% NaCl: 1.022 ± 0.001
9% Sucrose: 1.034 ± 0.001
RI calculation
light velocity of air/ light velocity solution
True or false: Specimens with very high SG readings can be diluted and retested
True
Reagent Strip Reaction for Specific Gravity: Principle
Change in pKa (dissociation constant of a polyelectrolyte)
Reagent Strip Reaction for Specific Gravity: Reagents
Multistix: Poly (methyl vinyl ether/ maleic anhydride) bromthymol blue
Chemstrip: Ethyleneglycoldiaminoethylethertetraacetic acid bromthymol blue
Reagent Strip Reaction for Specific Gravity: Interferences
False (+):
False (-):
False (+): high concentration of protein
False (-): Highly alkaline urines (>6.5)
Urine: Low Specific Gravity
specimen not concentrated, kidney disease.
Urine: High Specific Gravity
first morning, certain drugs
Urine: Random specimen SG
(1.003 to 1.032)
Urine: 24 hours specimen SG
(1.015 to 1.025)
Severe renal tubular damage, sp. Gravity is fixed at
1.010 =isothenuric
Based on frequency of soundwave entering a solution changes in proportion to the density of the solution
Harmonic Oscillation Densitometry
Example of Harmonic Oscillation Densitometry
Yellow IRIS (International Remote Imaging System)
Harmonic Oscillation Densitometry
Urine volume:
For IRIS Slideless microscope:
For IRIS Mass Gravity Meter:
Urine volume: 6 mL
For IRIS Slideless microscope: 4 mL
For IRIS Mass Gravity Meter: 2 mL
Important in the identification of crystals and determination of unsatisfactory specimens
pH
Normal pH
o Random:
o 1st morning:
o pH of 9.0-
o Random: 4.5- 8.0
o 1st morning: 5.0- 6.0
o pH of 9.0- unpreserved urine
Causes of Acid Urine (8)
Diabetes Mellitus (Inc. Ketone bodies)
Starvation (Inc. Ketone bodies)
High protein diet
Cranberry juice (Tx for UTI)
Emphysema
dehydration
diarrhea
presence of acid-producing bacteria (E.coli)
Causes of Alkaline Urine (7)
Renal tubular acidosis
Vegetarian diet
After meal- due to alkaline tide (withdrawal of H ions)
Vomiting
Old specimens
hyperventilation
presence of urease-producing bacteria
Reagent Strip Reaction for pH (60 secs): principle
Double indicator system
Reagent Strip Reaction for pH (60 secs): Reagents
Methy Red
Bromthymol Blue
Reagent Strip Reaction for pH (60 secs) : Interferences
No known interfering substances
Correlate: Nitrite, Leukocytes, Microscopic
Most indicative of renal disease
Protein
Indication of presence of protein in urine
Presence of white foam when shaken
Major serum protein found in the urine
Albumin
Albumin
Major serum protein found in the urine
Normal values:
Henry:
Stras:
Henry:<150 mg/day
Stras:<100 mg/day or <10 mg/dL
Other proteins beside from albumin
• Serum and tubular microglobulins
• Tamm-Horsfall protein (Uromodulin)
• Protein derived from prostatic and vaginal secretion
Caused by conditions that affect the plasma prior to it reaching the kidney
PRE-RENAL (“Before”) or Proteinuria (Overflow Proteinuria)
PRE-RENAL (“Before”) or Proteinuria (Overflow Proteinuria)
Intravascular Hemolysis:
Muscle injury:
Severe infection/Inflammation:
Multiple Myeloma:
Intravascular Hemolysis: Increase Hemoglobin
Muscle injury: increase Myoglobin
Severe infection/Inflammation: increase acute phase reactants
Multiple Myeloma: Proliferation of Ig producing plasma cells= BENCE-JONES PROTEIN
Tests for Bence-Jones Protein (2)
o Serum electrophoresis
o Immunofixation electrophoresis
Bence-Jones Protein
In Urine:
Precipitates @ ? –> ?
Dissolves @ ? (?)
In Urine:
Precipitates @ 40-60 °C –> producing cloudy urine Dissolves @ 100 °C (clear urine)
A. Glomerular Proteinuria
1. Diabetic Nephropathy
-Decrease _____
-May lead to ______
Indicator: _______
-Decrease GFR
-May lead to renal failure
Indicator: Microalbuminuria (Proteinuria undetectable by routine reagent strip)
Proteinuria undetectable by routine reagent strip
Microalbuminuria
Micral Test:
Principle:
Reading time:
False (-):
Result: (+) ____, (-) ____
Micral Test:
Principle: Enzyme immunoassay
Reading time: 60 seconds/1 minute
False (-): diluted urine
Result: (+) Red, (-) white
Albumin Excretion Rate (AER)= in ug/min or in mg/24 hrs
Normal AER:
Microalbuminuria:
Clinical albuminuria:
Normal AER: 0-20 ug/min
Microalbuminuria: 20-200 ug/min
Clinical albuminuria: >200 ug/min
Orthostatic Proteinuria is also known as
Cadet/ Postural Proteinuria
Proteinuria when standing due to increased pressure to renal veins
Orthostatic Proteinuria
ORTHOSTATIC PROTEINURIA
FIRST MORNING:
2 HOURS AFTER STANDING:
FIRST MORNING: -
2 HOURS AFTER STANDING: +
CLINICAL PROTEINURIA
FIRST MORNING:
2 HOURS AFTER STANDING:
FIRST MORNING: +
2 HOURS AFTER STANDING: +
Normally filtered albumin can no longer be reabsorbed
TUBULAR PROTEINURIA
TUBULAR PROTEINURIA disorders: (3)
• Fanconi’s syndrome
• Toxic agents/ heavy metals
• Severe viral infections
Post-renal proteinuria (After) (5)
- Lower UTI/ Inflammation
- Injury/ trauma
- Menstrual contamination
- Prostatic fluid/ spermatozoa
- Vaginal secretions
Reagent Strip Reaction for Protein (60 secs) Principle
Protein (Sorensen’s) error of indicator
Reagent Strip Reaction for Protein (60 secs): Reagents
Multistix: Tetrabromphenol blue
Chemstrip: Tetrachlorophenol tetrabromosulfonphthalein
Reagent Strip Reaction for Protein (60 secs): Interferences
False (+): high SG, highly buffered alkaline urine, pigmented specimen, antiseptics, phenazopyridine, detergents
False (-): protein other than albumin, microalbuminuria
Reagent Strip Reaction for Protein (60 secs): Remark
Indicator is sensitive to Albumin
Correlated to blood, nitrite, leukocytes, microscopic
A cold precipitation test that reacts equally with all forms of protein
Sulfosalicylic Acid (SSA) Precipitation Test
Reporting SSA Turbidity
Turbidity: Protein Range
(mg/dL)
Negative:
Trace:
1+
2+
3+
4+
Negative: no increase in turbidity; less than 6
Trace: Noticeable turbidity; 6-30
1+: Distinct Turbidity, no granulation; 30-100
2+: turbidity, granulation, no flocculation; 100-200
3+: turbidity, granulation, flocculation; 200-400,
4+: clump of protein; >400
Most frequently tested in urine
Glucose (Dextrose)
Renal threshold for glucose
160-180 mg/dL (plasma concentration of a subs. At which tubular reabsorption stops
Increase blood glucose= increase urine glucose
Hyperglycemia-associated
Causes of hyperglycemia
DM
Cushing’s syndrome (inc cortisol)
Pheochromocytoma (inc catecholamines)
Acromegaly (inc growth hormone)
Hyperthyroidism (inc T3, T4)
Normal glucose=increase urine glucose
Renal-associated
Cause of Renal-associated
Defective tubular reabsorption of glucose and amino acids
Reagent Strip Reaction for Glucose (30 secs): principle
Double sequential Enzyme Reaction
Reagent Strip Reaction for Glucose (30 secs): reagents
Multistix: Glucose oxidase, Peroxidase, Potassium iodide (blue to green to brown) Chemstrip: Glucose oxidase, Peroxidase, Tetramethylbenzidine (yellow to green)
Reagent Strip Reaction for Glucose (30 secs): interferences
False (+): Oxidizing agents, detergents
False (-): Ascorbic acid, ketones, high SG, Low temperature, improper preserve urine
Reagent Strip Reaction for Glucose (30 secs): remarks
Correlated: ketones, proteins