Standardization of Urinalysis
SPECIMEN COLLECTION FOR ROUTINE URINALYSIS
– 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
Collection of urine is started in the
morning and subsequent samples are
collected till the next morning.
Preservatives used:
Refrigeration at 4 degrees
Toluene- 1 ml / 50ml of urine. Preserves
chemical constituents.
SPECIMEN COLLECTION FOR 24 HOURS URINALYSIS
Thymol – 1% of thymol, gives false positive for proteins
Acid- HCl, sulfuric acid and boric acid can be used.
Formalin- 6-8 drops of 40% formalin per 100 ml
of urine. Preserves RBCs and Pus cells, gives
false positive test for sugar.
Specimen Collection
Supra-pubic Needle Aspiration
− Macroscopic Examination
− Chemical Analysis
− Microscopic Examination
− Culture
− Cytological Examination
Types of Analysis
Macroscopic Examination
Volume : normal – 700-2500ml/24 hours
Polyuria- >2500ml
Excess intake
Diabetes insipidus
Diabetes mellitus
Oliguria: <500ml
Less intake
Dehydration
Renal ischaemia
Anuria: <150ml
Renal stones
Renal or prostatic tumors
Severe renal ischaemia
Appearance normal-clear
Cloudy
U.T.I.
calculi
dye
Milky
lipiduria
Chyluria
Turbid
Crystals
infection
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)
Foam If a normal urine specimen is shaken or
agitated sufficiently, a white foam can be forced to develop at its surface that readily dissipates on standing.
Moderate to large amounts of protein (albumin) in urine cause a stable white foam to be produced when the urine is poured or agitated.
When bilirubin is present in sufficient amounts, the foam if present will be characteristically yellow.
Chemical Analysis
Conventional tests
Benedicts test
Heat coagulation test
Rothera’s test
Hay’s test
Ehrlich’s test
Fouchet’s test
Benzidine test
Chemical AnalysisUrine Dipstick
Glucose
Bilirubin
Ketones
Specific Gravity
Blood
pH
Protein
Urobilinogen
Nitrite
Leukocyte Esterase
Negative
Trace (100 mg/dL)
+ (250 mg/dL)
++ (500 mg/dL)
+++ (1000 mg/dL)
++++ (2000+ mg/dL)
The Urine Dipstick:Glucose
Glucose + 2 H2O + O2 --->Gluconic Acid + 2 H2O2
Glucose Oxidase
3 H2O2 + KI ---> KIO3 + 3 H2O
Horseradish Peroxidase
Chemical Principle
Read at 30 seconds
RR: Negative
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.
Uses and Limitations of Urine Glucose Detection
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
Detection of Reducing Sugars* by CuSO4
++
+
trace
400 600 800 1000200
Urin
aly
sis
Glu
co
se R
esu
lt
Blood Glucose (mg/dL)
Urine versus Blood Glucose
Negative
Negative
+ (weak)
++ (moderate)
+++ (strong)
The Urine Dipstick:Bilirubin
Bilirubin + Diazo salt ---------> AzobilirubinAcidic
Chemical Principle
Read at 30 seconds
RR: Negative
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
Uses and Limitations of Urine Bilirrubin Detection
Negative
Trace (5 mg/dL)
+ (15 mg/dL)
++ (40 mg/dL)
+++ (80 mg/dL)
++++ (160+ mg/dL)
The Urine Dipstick:Ketones
Acetoacetic Acid + Nitroprusside
------> Colored Complex
Chemical Principle
Read at 40 seconds
RR: Negative
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 version of same assay)
- Serum glucose measurement to confirm DKA
Uses and Limitations of Urine Ketone Detection
1.000
1.005
1.010
1.015
1.020
1.025
1.030
Chemical Principle
Read up to 2 minutes
RR: 1.003-1.035
The Urine Dipstick:Specific Gravity
pKa change of anpolyelectrolyte;indirect measureof density
Significance
- Diabetes insipidus
Limitations
- Interference: alkaline urine
- Does not measure non-ionized solutes (e.g. glucose)
Other Tests
- Refractometry
- Hydrometer
- Osmolality measurement
Uses and Limitations of Urine Specific Gravity
Negative
Trace (non-hemolyzed)
Moderate (non-hemolyzed)
Trace (hemolyzed)
+ (weak)
++ (moderate)
+++ (strong)
The Urine Dipstick:Blood
Diisopropylbenzene dihydroperoxide +
Tetramethylbenzidine
------------> Colored ComplexHeme
Chemical Principle
Lysing agent to lyse red blood cells
Read at 60 seconds
RR: Negative
Analytic Sensitivity: 10 RBCs
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
Uses and Limitations of Urine Blood Detection
5.0
6.0
6.5
7.0
7.5
8.0
8.5
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.: 4.5-8.0
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
Uses and Limitations of Urine pH Detection
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.
pH Run Over Effect
Negative
Trace
+ (30 mg/dL)
++ (100 mg/dL)
+++ (300 mg/dL)
++++ (2000 mg/dL)
The Urine Dipstick:Protein
Chemical Principle
H
H
H
H
H
H
Pr
Pr
Pr
Pr
Pr
Pr
“Protein Error of Indicators Method”
Pr Pr
Pr
PrPr
Pr
Tetrabromphenol Blue
(buffered to pH 3.0)H+
H+
H+H+
H+ H+
Read at 60 seconds
RR: Negative
Physiological 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
Causes of Proteinuria
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
Nephrotic Syndrome (> 3.5 g/dL in 24 h)
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)
Uses and Limitations of Urine Protein Detection
0.2 mg/dL
1 mg/dL
2 mg/dL
4 mg/dL
8 mg/dL
The Urine Dipstick:Urobilinogen
Urobilinogen + Diethylaminobenzaldehyde
-------> Colored Complex
(Ehrlich’s Reagent)
Chemical Principle
Read at 60 seconds
RR: 0.02-1.0 mg/dL
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
Uses and Limitations of Urobilinogen Detection
Reading time in a nutshell
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)
Microscopic ExaminationGeneral Aspects
Microscopic Examination
Per High Power Field (HPF) (400x)– > 3 erythrocytes
– > 5 leukocytes
– > 2 renal tubular cells
– > 10 bacteria
Per Low Power Field (LPF) (200x)– > 3 hyaline casts or > 1 granular cast
– > 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
Abnormal Findings
Erythrocytes- “Dysmorphic” vs. “normal” (> 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
Microscopic ExaminationCells
Microscopic ExaminationRBCs
Microscopic ExaminationWBCs
Microscopic ExaminationSquamous Cells
Microscopic ExaminationTubular Epithelial Cells
Microscopic ExaminationTransitional Cells
Microscopic ExaminationTransitional Cells
Microscopic ExaminationOval Fat Body
Bacteria- Bacteriuria More than 10 per HPF
Yeasts- Candidiasis Most likely a contaminant
but should correlate withclinical picture.
Viruses- CMV inclusions Probable viral cystitis.
Microscopic ExaminationBacteria & Yeasts
Microscopic ExaminationBacteria
Microscopic ExaminationYeasts
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)
Microscopic ExaminationCasts
Microscopic ExaminationCasts
Microscopic ExaminationRBCs Cast - Histology
Microscopic ExaminationRBCs Cast
Microscopic ExaminationRBCs Cast - Histology
Microscopic ExaminationWBCs Cast
Microscopic ExaminationTubular Epith. Cast
Microscopic ExaminationTubular Epith. Cast
Microscopic ExaminationGranular Cast
Microscopic ExaminationHyaline Cast
Microscopic ExaminationWaxy Cast
Microscopic ExaminationFatty Cast
Bacterial Casts
Single Leukocytes
Leukocyte Casts
Verrier-Jones & Asscher, 1991.
Single Erythrocytes
Erythrocyte Casts
Single Bacteria
Significance of Cellular Casts
Microscopic ExaminationCrystals in acidic urine
Microscopic ExaminationCrystals in alkaline urine
Microscopic ExaminationCalcium Oxalate Crystals
Microscopic ExaminationCalcium Oxalate Crystals
Dumbbell Shape
Microscopic ExaminationTriple Phosphate Crystals
Microscopic ExaminationUrate Crystals
Microscopic ExaminationLeucine Crystals
Microscopic ExaminationCystine Crystals
Microscopic ExaminationAmmonium Biurate Crystals
Microscopic ExaminationCholesterol Crystals
Need for automation
Time saving
Easy to process
Multiple samples can be processed
Reduce the need for man power
Uniformity
Sensitive
Specific
List of instruments
Principle
Aspirates and dispenses fixed amount of urine on each pad of the strip
Reads the strip via reflectance method.
Assess color of a specimen by using four wavelengths of light to obtain the tone (light, normal, dark) and hue of a urine specimen.
Light scatter is used to determine the turbidity
Specific gravity is measured by assessing refractive index of LED-emitted light.
dual wavelength reflectance to measure the pH and chemical constituents of urine
Priciple
Iris Diagnostics Division iChem®100
Factors That Require Standardization in the Microscopic Examination Urine volume used (e.g., 10 mL, 12 mL, 15 mL)
Speed of centrifugation (400 or 450 Å~ g)
Time of centrifugation (5 minutes)
Concentration of sediment prepared (e.g., 10 : 1, 12 : 1, 15 : 1, 30 : 1)
Volume of sediment examined—determined by commercial slides used and microscope optical properties (i.e., ocular field number)
Result reporting—format, terminology, reference intervals, magnification used for assessment
video
Cytological Examination
Staining:
Papanicolau
Wright’s
Immunoperoxidase
Immunofluorescence
Cytology: Normal
Cytology: Normal
Cytology: Reactive
Cytology: Reactive
Cytology: Polyoma (Decoy Cell)
Cytology: Polyoma (Decoy Cell)Immunoperoxidase to SV40 ag
Cytology: TCC Low Grade
Cytology: TCC Low Grade
Cytology: TCC High Grade
Cytology: TCC High Grade
Cytology: Squamous Cell Ca.
Cytology: Renal Cell Ca.
Cytology: Prostatic Carcinoma
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