Urinalysis

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Urinalysis
Course IDPT 5005 School of Medicine, UCDHSC
Francisco G. La Rosa, MD Francisco.LaRosa_at_uchsc.e
du Assistant Professor, Department of
Pathology University of Colorado at Denver Health
Science Center, Denver, Colorado
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Specimen Collection

• First morning voiding (most concentrated)
• Record collection time
• Type of specimen (e.g. clean catch)
• Analyzed within 2 hours of collection
• Free of debris or vaginal secretions

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Clean Catch
4
Specimen Collection
Supra-pubic Needle Aspiration
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Types of Analysis

• Macroscopic Examination
• Chemical Analysis (Urine Dipstick)
• Microscopic Examination
• Culture (not covered in this lecture)
• Cytological Examination

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Macroscopic Examination

• Odor
• Ammonia-like (Urea-splitting bacteria)
• Foul, offensive Old specimen, pus or
  inflammation
• Sweet Glucose
• Fruity Ketones
• Maple syrup-like Maple Syrup Urine Disease

• Color
• Colorless Diluted urine
• Deep Yellow Concentrated Urine, Riboflavin
• Yellow-Green Bilirubin / Biliverdin
• Red Blood / Hemoglobin
• Brownish-red Acidified Blood (Actute GN)
• Brownish-black Homogentisic acid (Melanin)

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Macroscopic Examination

• Turbidity
• Typically cells or crystals.
• Cellular elements and bacteria will clear by
  centrifugation.
• Crystals dissolved by a variety of methods (acid
  or base).
• Microscopic examination will determine which is
  present.

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Chemical Analysis
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Chemical Analysis
Urine Dipstick
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The Urine Dipstick
Glucose
Chemical Principle
Glucose Oxidase
Glucose 2 H2O O2 ---gt Gluconic Acid 2 H2O2
Horseradish Peroxidase
3 H2O2 KI ---gt KIO3 3 H2O
Read at 30 seconds RR Negative
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Uses and Limitations of Urine Glucose Detection

• Significance
• Diabetes mellitus.
• Renal glycosuria.
• Limitations
• Interference reducing agents, ketones.
• Only measures glucose and not other sugars.
• Renal threshold must be passed in order for
  glucose to spill into the urine.
• Other Tests
• CuSO4 test for reducing sugars.

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Detection of Reducing Sugars by CuSO4

• Sugar Disease(s)
• - Galactose Galactosemias
• - Fructose Fructosuria, Fructose Intolerance,
  etc.
• - Lactose Lactase Deficiency
• - Pentoses Essential Pentosuria
• - Maltose Non-pathogenic
• NOT Sucrose because it is not a reducing sugar

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Urine versus Blood Glucose


Urinalysis Glucose Result
trace
Negative
400
600
800
1000
200
Blood Glucose (mg/dL)
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The Urine Dipstick
Bilirrubin
Chemical Principle
Acidic
Bilirubin Diazo salt ---------gt Azobilirubin
Read at 30 seconds RR Negative
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Uses and Limitations of Urine Bilirrubin Detection

• Significance
• - Increased direct bilirubin (correlates with
  urobilinogen and serum bilirubin)
• Limitations
• - Interference prolonged exposure of sample to
  light
• - Only measures direct bilirubin--will not pick
  up indirect bilirubin
• Other Tests
• - Ictotest (more sensitive tablet version of
  same assay)
• - Serum test for total and direct bilirubin is
  more informative

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The Urine Dipstick
Ketones
Chemical Principle
Acetoacetic Acid Nitroprusside ------gt Colored
Complex
Read at 40 seconds RR Negative
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Uses and Limitations of Urine Ketone Detection

• Significance
• - Diabetic ketoacidosis
• - Prolonged fasting
• Limitations
• - Interference expired reagents (degradation
  with exposure to moisture in air)
• - Only measures acetoacetate not other ketone
  bodies (such as in rebound ketosis).
• Other Tests
• - Ketostix (more sensitive tablet version of
  same assay)
• - Serum glucose measurement to confirm DKA

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The Urine Dipstick
Specific Gravity
Chemical Principle
X Polymethyl vinyl ether / maleic
anhydride ---------------gt 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 1.003-1.035
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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)

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

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The Urine Dipstick
pH
Chemical Principle
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)
Read up to 2 minutes R.R. 4.5-8.0
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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 (gt5.5)
• 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

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pH Run Over Effect
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.
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The Urine Dipstick
Protein
Chemical Principle
Protein Error of Indicators Method
Tetrabromphenol Blue (buffered to pH 3.0)
Pr
Pr
Pr
Pr
Pr
Pr
Read at 60 seconds RR Negative
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Causes of Proteinuria

• Functional Renal
• - Severe muscular exertion - Glomerulonephritis
• - Pregnancy - Nephrotic syndrome
• - Orthostatic proteinuria - Renal tumor or
  infection
• Pre-Renal Post-Renal
• - Fever - Cystitis
• - Renal hypoxia - Urethritis or prostatitis
• - Hypertension - Contamination with vaginal
  secretions

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Nephrotic Syndrome (gt 3.5 g/dL in 24 h)

• Primary
• - Lipoid nephrosis (severe)
• - Membranous glomerulonephritis
• - Membranoproliferative glomerulonephritis
• Secondary
• - Diabetes mellitus (Kimmelsteil-Wilson lesions)
• - Systemic lupus erythematosus
• - Amyloidosis and other infiltrative diseases
• - Renal vein thrombosis

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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

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Proteins in Normal Urine

• Protein of Total Daily Maximum
• Albumin 40 60 mg
• Tamm-Horsfall 40 60 mg
• Immunoglobulins 12 24 mg
• Secretory IgA 3 6 mg
• Other 5 10 mg
• TOTAL 100 150 mg

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The Urine Dipstick
Urobilinogen
Chemical Principle
Urobilinogen Diethylaminobenzaldehyde -------gt
Colored Complex
(Ehrlichs Reagent)
Read at 60 seconds RR 0.02-1.0 mg/dL
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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 ---gt urobilin)
• - Cannot detect low levels of urobilinogen
• Other Tests
• - Serum total and direct bilirubin

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The Urine Dipstick
Nitrite
Chemical Principle
Acidic
Nitrite p-arsenilic acid -------gt Diazo compound
Diazo compound Tetrahydrobenzoquinolinol -------
---gt Colored Complex
Read at 60 seconds RR Negative
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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

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The Urine Dipstick
Leukocyte Esterase
Chemical Principle
Derivatized pyrrole amino acid ester
------------gt 3-hydroxy-5-phenyl pyrrole
Esterases
3-hydroxy-5-phenyl pyrrole diazo
salt -------------gt Colored Complex
Read at 2 minutes RR Negative Analytic
Sensitivity 3-5 WBCs
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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

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Microscopic Examination
General Aspects

• Preservation
• - Cells and casts begin to disintegrate in 1 - 3
  hrs. at room temp.
• - Refrigeration for up to 48 hours (little loss
  of cells).
• Specimen concentration
• - Ten to twenty-fold concentration by
  centrifugation.
• Types of microscopy
• - Phase contrast microscopy
• - Polarized microscopy
• - Bright field microscopy with special staining
• (e.g., Sternheimer-Malbin stain)

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Microscopic Examination
Abnormal Findings

• Per High Power Field (HPF) (400x)
• gt 3 erythrocytes
• gt 5 leukocytes
• gt 2 renal tubular cells
• gt 10 bacteria
• Per Low Power Field (LPF) (200x)
• gt 3 hyaline casts or gt 1 granular cast
• gt 10 squamous cells (indicative of contaminated
  specimen)
• Any other cast (RBCs, WBCs)
• Presence of
• Fungal hyphae or yeast, parasite, viral
  inclusions
• Pathological crystals (cystine, leucine,
  tyrosine)
• Large number of uric acid or calcium oxalate
  crystals

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Microscopic Examination
Cells

• Erythrocytes
• - Dysmorphic vs. normal (gt 10 per HPF)
• Leukocytes
• - Neutrophils (glitter cells) More than 1 per 3
  HPF
• - Eosinophils Hansel test (special stain)
• Epithelial Cells
• - Squamous cells Indicate level of contamination
• - Renal tubular epithelial cells Few are normal
• - Transitional epithelial cells Few are normal
• - Oval fat bodies Abnormal, indicate Nephrosis

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Microscopic Examination
RBCs
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Microscopic Examination
RBCs
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Microscopic Examination
WBCs
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Microscopic Examination
Squamous Cells
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Microscopic Examination
Tubular Epithelial Cells
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Microscopic Examination
Transitional Cells
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Microscopic Examination
Transitional Cells
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Microscopic Examination
Oval Fat Body
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Microscopic Examination
LE Cell
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Microscopic Examination
Bacteria Yeasts

• Bacteria
• - Bacteriuria More than 10 per HPF
• Yeasts
• - Candidiasis Most likely a contaminant
• but should correlate with
• clinical picture.
• Viruses
• - CMV inclusions Probable viral cystitis.

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Microscopic Examination
Bacteria
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Microscopic Examination
Yeasts
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Microscopic Examination
Yeasts
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Microscopic Examination
Cytomegalovirus
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Microscopic Examination
Casts

• Erythrocyte Casts Glomerular diseases
• Leukocyte Casts Pyuria, glomerular disease
• Degenerating Casts
• - Granular casts Nonspecific (Tamm-Horsfall
  protein)
• - Hyaline casts Nonspecific (Tamm-Horsfall
  protein)
• - Waxy casts Nonspecific
• - Fatty casts Nephrotic syndrome
• (oval fat body casts)

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Microscopic Examination
Casts
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Microscopic Examination
RBCs Cast - Histology
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Microscopic Examination
RBCs Cast
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Microscopic Examination
RBCs Cast - Histology
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Microscopic Examination
WBCs Cast
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Microscopic Examination
Tubular Epith. Cast
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Microscopic Examination
Tubular Epith. Cast
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Microscopic Examination
Granular Cast
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Microscopic Examination
Hyaline Cast
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Microscopic Examination
Waxy Cast
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Microscopic Examination
Fatty Cast
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Significance of Cellular Casts
Erythrocyte Casts
Leukocyte Casts
Bacterial Casts
Single Erythrocytes
Single Leukocytes
Single Bacteria
Verrier-Jones Asscher, 1991.
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Microscopic Examination
Crystals

• - Urate
• Ammonium biurate
• Uric acid
• - Triple Phosphate
• - Calcium Oxalate
• - Amino Acids
• Cystine
• Leucine
• Tyrosine
• - Sulfonamide

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Microscopic Examination
Calcium Oxalate Crystals
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Microscopic Examination
Calcium Oxalate Crystals
Dumbbell Shape
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Microscopic Examination
Triple Phosphate Crystals
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Microscopic Examination
Urate Crystals
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Microscopic Examination
Leucine Crystals
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Microscopic Examination
Cystine Crystals
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Microscopic Examination
Ammonium Biurate Crystals
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Microscopic Examination
Cholesterol Crystals
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Cytological Examination

• Staining
• Papanicolau
• Wrights
• Immunoperoxidase
• Immunofluorescence

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Cytology Normal
77
Cytology Normal
78
Cytology Reactive
79
Cytology Reactive
80
Cytology Polyoma (Decoy Cell)
81
Cytology Polyoma (Decoy Cell)Immunoperoxidase
to SV40 ag
82
Cytology TCC Low Grade
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Cytology TCC Low Grade
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Cytology TCC High Grade
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Cytology TCC High Grade
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(No Transcript)
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(No Transcript)
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Cytology Squamous Cell Ca.
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Cytology Renal Cell Ca.
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Cytology Prostatic Carcinoma
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Disease Diagnosis
Urinalysis
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Diluted urine, request a voided urine in the
morningIf persisting low SG, possible diabetes
insipidaA microscopic may give negative results
Case 1
Negative
Glucose
A 35-year old man undergoing routine pre
employment drug screening. Physical
characteristics Clear. Microscopic Not
performed. Drugs Identified None. Questions -
What is your differential diagnosis? - What
would you do next to confirm your suspicion? -
Would you order a microscopic analysis on this
sample?
Negative
Bilirubin
Negative
Ketones
1.001
S.G.
Negative
Blood
5.5
pH
Negative
Protein
0.2 mg/dL
Urobilinogen
Negative
Nitrite
Negative
L.E.
93
Possible gallbladder or hepatic disease.No
hemolytic anemia. Perform bilirubins in
serumMicroscopic unlikely to provide additional
info
Case 2
Glucose
Negative
Bilirubin

A 42-year old woman presents with dark
urine Physical characteristics
Red-brown. Microscopic Not performed. Questions
- What is your differential diagnosis? - Could
this be a case of hemolytic anemia? - How would
you rule it out? - What tests would you order
next? Why? - Would you order a microscopic
analysis?
Ketones
Negative
S.G.
1.020
Blood
Negative
pH
5.5
Protein
Negative
Urobilinogen
0.2 mg/dL
Nitrite
Negative
L.E.
Negative
94
Possible UTI, request culture and antibiotic
sensitivityNegative Nitrite test Gram positive
bacteriaLower SG may show less number of cells
and bacteriaUn-common diagnosis in this type of
patient
Case 3
Glucose
Negative
A 42-year old man presents painful
urination Physical characteristics dark red,
turbid Microscopic leukocytes 30 per HPF RBCs
gt100 per HPF Bacteria gt100 per
HPF Questions - What is your suspected
diagnosis? - What would you do next? - What do
you make of the nitrite test? - How would the
microscopic exam differ if the S.G. were 1.003? -
Is this a common diagnosis for this type of
patient?
Bilirubin
Negative
Ketones
Negative
S.G.
1.030
Blood

pH
6.5
Protein
Trace
Urobilinogen
1.0 mg/dL
Nitrite
Negative
L.E.

95
DiabetesMay be decompensated and with
ketoacidosisKetones should become negative after
treatment
Case 4

Glucose
Bilirubin
Negative
A 27-year old woman presents with severe
abdominal pain. Physical characteristics
clear-yellow. Microscopic Not performed. Questio
ns - What is the most likely diagnosis? - What
do you make of the ketone result? - What do you
expect to happen to the ketone measurement when
treatment begins?
Ketones
Trace
1.015
S.G.
Negative
Blood
pH
6.0
Protein
Negative
1.0 mg/dL
Urobilinogen
Negative
Nitrite
L.E.
Negative
96
GlomerulonephritisRBC casts reveals renal cortex
involvementRBC cast are not always present in GN
Case 5
Glucose
Negative
8-year old boy presents with discolored
urine Physical characteristics Red,
turbid. Microscopic erythrocytes gt100 per HPF
(almost all dysmorphic) Red cell casts
present. Questions - What is the most likely
diagnosis in this case? - Does the presence of
red cell casts help you in any way? - If the
erythrocytes were not dysmorphic would that
change your diagnosis?
Bilirubin
Negative
Ketones
Negative
S.G.
1.015
Blood

pH
6.5
Protein

Urobilinogen
1.0 mg/dL
Nitrite
Negative
L.E.
Negative
97
Functional proteinuria?Microscopic may reveal
a few leukocytesRequest protein concentration in
24 h urine
Case 6
Negative
Glucose
22-year old man presenting for a routine physical
required for admission to medical
school Physical characteristics
Yellow Microscopic Not performed Questions -
What is your differential diagnosis? - Would you
order a microscopic analysis on this sample? -
What would you do next to confirm the diagnosis?
Negative
Bilirubin
Negative
Ketones
1.010
S.G.
Negative
Blood
5.0
pH

Protein
0.2 mg/dL
Urobilinogen
Negative
Nitrite
Negative
L.E.
98
Common Findings inAcute Tubular Necrosis
Glucose
Bilirubin
Ketones

• Microscopic
• Renal tubular epithelial cells
• Pathological casts

Decreased
S.G.
/ -
Blood
pH
/ -
Protein
Urobilinogen
Nitrite
L.E.
99
Common Findings inAcute Glomerulonephritis
Glucose
Bilirubin
Ketones

• Microscopic
• Erythrocytes (dysmorphic)
• Erythrocyte casts
• Mixed cellular casts

S.G.
Increased
Blood
pH
Increased
Protein
Urobilinogen
Nitrite
L.E.
100
Common Findings inChronic Glomerulonephritis
Glucose
Bilirubin
Ketones

• Microscopic
• Pathological casts(broad waxy casts, RBCs)

Decreased
S.G.
Increased
Blood
pH
Increased
Protein
Urobilinogen
Nitrite
L.E.
101
Common Findings inAcute Pyelonephritis
Glucose
Bilirubin

• Microscopic
• Bacteria
• Leukocytes
• Leukocyte, granular, andwaxy casts
• Renal tubular epithelialcell casts

Ketones
S.G.
Blood
pH
Protein
Trace
Urobilinogen
Positive
Nitrite
Positive
L.E.
102
Common Findings inNephrotic Syndrome
Glucose
Bilirubin
Ketones

• Microscopic
• Oval fat bodies
• Fatty casts
• Waxy casts

S.G.
Blood
pH

Protein
Urobilinogen
Nitrite
L.E.
103
Common Findings inEosinophilic Cystitis
Glucose
Bilirubin
Ketones

• Microscopic
• Numerous eosinophils (Hansels stain)
• NO significant casts.

S.G.

Blood
pH
Protein
Urobilinogen
Nitrite
L.E.
104
Common Findings inUrothelial Carcinoma
Glucose
Bilirubin
Ketones

• Microscopic
• Malignant cells on urine cytology (urine sample
  should be submitted separately to cytology, void
  or 24 hrs.)

S.G.

Blood
pH
Protein
Urobilinogen
Nitrite
L.E.
105
Acknowledgment Dr. Brad Brimhall
106
Questions ?