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Urine analysis
Dr. Ahmadvand
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Introduction Urine is formed in the kidneys, is a product
of ultrafiltration of plasma by the renal glomeruli.
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Formation of Urine
Three processes of urine formation: glomerular filtration tubular reabsorption tubular secretion
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Physical Composition and Chemical Properties Urine 95% water5% waste products
Urea, uric acid, ammonia, calcium, creatine, sodium, chloride, potassium, sulfates, phosphates, bicarbonates, hydrogen ions, urochrome, urobilinogen
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Collection of urine
Early morning sample-qualitative Random sample- routine 24hrs sample- quantitative Midstream sample
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24 hour urine sample
1. For quantitative estimation of proteins
2. For estimation of vanillyl mandelic acid, 5-hydroxyindole acetic acid, metanephrines
3. For detection of microalbuminuria
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Preservation and StorageChanges that affect
the chemical or microscopic properties of urine occur if urine is kept at room temperature for more than 1 hour
Refrigeration – most common method for storing and preserving urine
It prevents bacterial growth for 24 hours.
After 24 hours use chemical preservation
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Urinalysis
Evaluation of urine to obtain information about body health and disease
Three types of testing: Physical Chemical Microscopic
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Urine examination
Macroscopic examination Chemical examination Microscopic examination
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Macroscopic examination
Volume Color Odour Urinary pH Specific gravity
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Physical Examination of UrineVisual examination of
physical characteristics Color and turbidity Volume Odor Specific gravity
The refractometer or a reagent strip is used to measure specific gravity
Reagent Strips
Refractometer
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Normal Values of Urine Normal values of various
elements have been established
Average adult daily urine output is 1250 mL/24 hours
Intake and output should be approximately the same
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Urinary volume
Normal = 600-1550ml Polyuria- >2000ml Oliguria-<400ml Anuria-complete cessation of
urine(<200ml)
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Causes of polyuria
Diabetes mellitus Diabetes insipidus Diuretics Intravenous saline/glucose
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oliguria
Dehydration-vomiting, diarrhoea, excessive sweating
Renal ischemia Acute tubular necrosis Obstruction to the urinary tract
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Color & appearance
Normal= clear & pale yellow
1. Colourless- dilution, diabetes mellitus, diabetes insipidus, diuretics
2. Milky-purulent genitourinary tract infection
3. Orange- excessive sweating
4. Red-beetroot ingestion,haematuria
5. Brown/ black- alkaptunuria, melanin
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Urinary pH/ reaction
Reaction reflects ability of kidney to maintain normal hydrogen ion concentration in plasma
Normal= 4.6-8 Tested by- 1.litmus paper
2. pH paper
3. dipsticks
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Acidic urine
Ketosis-diabetes, starvation, fever Acidosis Acidification therapy
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Alkaline urine
Strict vegetarian Systemic alkalosis Alkalization therapy
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Odour
Normal= aromatic due to the volatile fatty acids
Ammonical – bacterial action Fruity- ketonuria
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Specific gravity
Depends on the concentration of various solutes in the urine.
Measured by-
- refractometer
- dipsticks
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High specific gravity
Normal-1.016-1.022 Causes
All causes of oliguria
Gycosuria
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Low specific gravity
All causes of polyuria except gycosuria
Fixed specific gravity (isosthenuria)=1.010
Seen in chronic renal disease when kidney has lost the ability to concentrate or dilute
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Apply Your Knowledge
What is the specific gravity shown on this refractometer screen?
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Apply Your Knowledge -AnswerAnswer
The specific gravity shown here is 1.030
What is the specific gravity shown on this refractometer screen?
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Specific Gravity
Range = 1.016 and 1.022 Elevated specific gravity seen with
elevated protein levels (1 to 7.5 g/L)
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pH
Range from pH 5 to 8.5. False results may occur if excessive urine
remains on the strip.
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Nitrite
Dependant on the conversion of dietary nitrate to nitrite by Gram Negative Bacteria.
Positive results may indicate presence of greater than 105 cells per mL.
Color not proportional to the number of cells.
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Typical Test StripTest
Glucose
Bilirubin
Ketone
Blood
Protein
Nitrite
Leukocytes
pH
Specific Gravity
Urobilinogen
Sensitivity_ – 4 to 7 mmol/L– 7 to 14 mol/L– 0.5 to 1.0 mmol/L (Acetoacetic acid)– 150 to 620 g/L (Hemoglobin)– 0.15 to 0.3 g/L (Albumin)– 13 to 22 mol/L– 5 to 15 cells/ L– pH 5.0 to 8.5– 1.000 to1.030– 0.2 to 8 mol/L
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Specific Gravity
Range = 1.000 and 1.030 Elevated specific gravity seen with
elevated protein levels (1 to 7.5 g/L)
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Chemicals Found in Urine Ketone bodiesKetone bodies – may indicate that patient
is following a low carbohydrate diet or that patient has conditions such as starvation
pHpH – provides information about metabolic status, diet, medication or several conditions
Blood (hematuria)Blood (hematuria) – may indicate patient is menstruating, have urinary tract infection or trauma
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Chemicals Found in Urine (cont.)Bilirubin or urobilinogenBilirubin or urobilinogen – first signs of
liver disease
GlucoseGlucose – common in patients with diabetes
ProteinProtein – usually indicates renal disease
NitriteNitrite – suggests bacterial infection
Leukocytes Leukocytes – urinary or renal infection
PhenylketonesPhenylketones – indicates PKU
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Chemical Testing of Urine You may be asked to perform chemical tests
on urine Check for proper identification of urine
specimen Usually done with reagent strips or tablets Used to determine body processes such as
carbohydrate metabolism, liver or kidney function or acid-base balance.
Used to determine presence of drug, toxic environmental substances or infections
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Reagent Test Strips
Multiple test reagents on a ready to use test strip.
Test results are read at different times after brief exposure to urine sample.
The color on strip is compared to the reference color shown on test strip packaging.
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Glucose
Small amounts of glucose normally excreted by the kidney.
Below sensitivity of the test. >6 mmol/L clinically significant.
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Test for sugar Test-BENEDICT’S TEST(semiquantitative) Principle-benedict’s reagent contains cuso4.In the
presence of reducing sugars cupric ions are converted to cuprous oxide which is hastened by heating, to give the color.
Method- take 5ml of benedict’s reagent in a test tube, add 8drops of urine. Boil the mixture.
Blue-green= negative Yellow-green=+(<0.5%) Greenish yellow=++(0.5-1%) Yellow=+++(1-2%) Brick red=++++(>2%)
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Benedict’s test
Detects all reducing substances like glucose, fructose, & other reducing sustances.
To confirm it is glucose, dipsticks can be used (glucose oxidase)
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Causes of glycosuria
Glycosuria with hyperglycaemia- diabetes,acromegaly, cushing’s disease, hyperthyroidism, drugs like corticosteroids.
Glycosuria without hyperglycaemia- renal tubular dysfunction
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Ketones
Test detects acetoacetic acid in urine. Normal urine negative with this test. Detectable levels seen during physiological
stress such as fasting, pregnancy, and frequent strenuous exercise.
Large amounts with ketoacidosis due to starvation and abnormal carbohydrate or lipid metabolism.
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Ketone bodies
3 types Acetone Acetoacetic acid β-hydroxy butyric acid
They are products of fat metabolism
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Rothera’s test
Principle-acetone & acetoacetic acid react with sodium nitroprusside in the presence of alkali to produce purple colour.
Method- take 5ml of urine in a test tube & saturate it with ammonium sulphate. Then add one crystal of sodium nitroprusside. Then gently add 0.5ml of liquor ammonia along the sides of the test tube.
Change in colour indicates + test
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Causes of ketonuria
Diabetes Non-diabetic causes- high fever,
starvation, severe vomiting/diarrhoea
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pH
Range from pH 5 to 8.5. False results may occur if excessive urine
remains on the strip.
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Protein
Test sensitive to albumin. Negative result does not rule out the
presence of hemoglobin or globulins. Normally no protein present in urine. Greater than 0.3 g/L is clinically
significant.
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Tests for proteins
Test – HEAT & ACETIC ACID TEST Principle-proteins are denatured & coagulated on
heating to give white cloud precipitate. Method-take 2/3 of test tube with urine, heat only
the upper part keeping lower part as control. Presence of phosphates, carbonates, proteins
gives a white cloud formation. Add acetic acid 1-2 drops, if the cloud persists it indicates it is protein(acetic acid dissolves the carbonates/phosphates)
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Other tests
Sulphosalicylic acid test Dipsticks Esbach’s albuminometer- for quantitative
estimation of proteins
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Causes of proteinuria
Prerenal causes-Heavy exercise,Fever,hypertension, multiple myeloma, ecalmpsia
Renal –acute & chronic glomerulonephritis,Renal tubular dysfunction,Polycystic kidney, nephrotic syndrome
Post renal- acute & chronic cystitis, tuberculosis cystitis
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microalbuminuria
The level of albumin protein produced by microalbuminuria cannot be detected by urine dipstick methods. In a properly functioning body, albumin is not normally present in urine because it is retained in the bloodstream by the kidneys. Microalbuminuria is diagnosed either from a 24-hour urine collection
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Significance of microalbuminuria
an indicator of subclinical cardiovascular disease an important prognostic marker for kidney disease in diabetes mellitus in hypertension increasing microalbuminuria during the first 48
hours after admission to an intensive care unit predicts elevated risk for acute respiratory failure , multiple organ failure , and overall mortality
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Bence Jones proteins
These are light chain globulins seen in multiple myeloma, macroglobulimias, lymphoma.
Test- Thermal method(waterbath):
Proteins has unusual property of precipitating at 400 -600c & then dissolving when the urine is brought to boiling(1000c) & reappears when the urine is cooled.
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Bilirubin
Normally no Bilirubin is detected in urine. Even trace amounts are clinically
significant.
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Bilirubin Test- fouchet’s test. Causes Liver diseases-injury,hepatitis Obstruction to biliary tract
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Urobilinogen
Normal Range in urine = 3 to 17 mol/L
Greater than 34 mol/L transition from normal to abnormal.
Total absence of urobilinogen cannot be determined with this test.
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Urobilinogen
Test- ehrlich test Causes-hemolytic anemia's
Bile salts-Hay’s testCause- obstruction to bile flow (obstructive
jaundice)
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Blood
Test measures hemoglobin and myoglobin. Trace amounts may be clinically significant. Often found in urine of menstruating
females. False positives may be seen with urinary
tract infections
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Blood in urine
Test- BENZIDINE TEST Principle-The peroxidase activity of hemoglobin
decomposes hydrogen peroxide releasing nascent oxygen which in turn oxidizes benzidine to give blue color.
Method- mix 2ml of benzidine solution with 2ml of hydrogen peroxide in a test tube. Take 2ml of urine & add 2ml of above mixture. A blue color indicates + reaction.
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Causes of hematuria Pre renal- bleeding diathesis,
hemoglobinopathies, malignant hypertension.
Renal- trauma, calculi, acute & chronic glomerulonephritis, renal TB, renal tumors
Post renal – severe UTI, calculi, trauma, tumors of urinary tract
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Type Plasma color Urine color
Hematuria normal Smoky red
m/s-plenty of RBC’s
hemoglobunuria Pink,hepatoglobin reduced
Red , occasional RBC’s
Myoglobunuria Pink, normal hepatoglobin
Red, occasional RBC’s
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BioMedica Diagnostics Inc. / D. Jette / March 2003
61
Nitrite
Dependant on the conversion of dietary nitrate to nitrite by Gram Negative Bacteria.
Positive results may indicate presence of greater than 105 cells per mL.
Color not proportional to the number of cells.
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Leukocytes
Normally no leukocytes present in urine. Positive result is clinically significant. Some drugs interfere with the test
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Urine dipsticks Urine dipstick is a narrow plastic strip which
has several squares of different colors attached to it. Each small square represents a component of the test used to interpret urinalysis. The entire strip is dipped in the urine sample and color changes in each square are noted. The color change takes place after several seconds to a few minutes from dipping the strip. If read too early or too long after the strip is dipped, the results may not be accurate.
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The main advantage of dipsticks is that they are
1. convenient,
2. easy to interpret,
3. and cost-effective
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The main disadvantage is that the1. Information may not be very accurate as
the test is time-sensitive.2. It also provides limited information about
the urine as it is qualitative test and not a quantitative test (for example, it does not give a precise measure of the quantity of abnormality).
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Microscopic Examination of Urine Microscopic examination used to view
elements not visible without microscope Centrifuge spins urine to cause heavier
substances to settle to the bottom
•Casts
•Yeasts
•Parasites
•Cells
•Crystals
•Bacteria
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Microscopic examination Microscopic urinalysis is done simply pouring
the urine sample into a test tube and centrifuging it (spinning it down in a machine) for a few minutes. The top liquid part (the supernatant) is discarded. The solid part left in the bottom of the test tube (the urine sediment) is mixed with the remaining drop of urine in the test tube and one drop is analyzed under a microscope
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Contents of normal urine m/s
Contains few epithelial cells, occasional RBC’s, few crystals.
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White blood cells/red blood cells in urine
This is a photograph of urinary sediment under brightfield microscopy (250X magnification). The sediment contains two red blood cells (right) and one white blood cell (left). The white blood cell appears to have a lobulated nucleus.
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Red blood cells in urine
These are red blood cells found in urinary sediment under bright field microscopy (160x magnification).
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WBC in Urine :
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White blood cells and bacteria
These are white blood cells and bacteria found in urinary sediment using interference-contrast microscopy. The cell in the center has a pseudopod extending from its cytoplasm indicating amoeboid motion.
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Squamous epithelial cells
Shown here is a group of squamous epithelial cells in urine sediment. Interference-contrast microscopy was used to enhance surface characteristics of the cells (200X magnification). A few white blood cells and bacteria are visible in the periphery.
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Squamous epithelial cell in urine
This is a squamous epithelial cell under brightfield microscopy (250X magnification).Squamous epithelial cells are characterized by a small, round, central nucleus with a large amount of granular or wrinkled cytoplasm (a "fried egg" appearance).
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Renal Epithelial Cells
A group of attached renal tubular epithelial cells are seen in the center of this slide. The background contains white blood cells and red blood cells (bright field microscopy, 250X magnification)
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casts
Urinary casts are cylindrical aggregations of particles that form in the distal nephron, dislodge, and pass into the urine. In urinalysis they indicate kidney disease. They form via precipitation of Tamm-Horsfall mucoprotein which is secreted by renal tubule cells.
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Urine Microscopy :
Cells Casts CrystalsCells Casts Crystals. Casts are formed within nephron. Casts Suggest Kidney pathology. Casts can be made up of Protein, lipid,
cells or mixed. Crystals suggest high concentration or
altered solubility.
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Types of casts
Acellular casts
Hyaline casts
Granular casts
Waxy casts
Fatty casts
Pigment casts
Crystal casts
Cellular casts
Red cell casts
White cell casts
Epithelial cell cast
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Hyaline casts
The most common type of cast, hyaline casts are solidified Tamm-Horsfall mucoprotein secreted from the tubular epithelial cells
Seen in fever, strenuous exercise, damage to the glomerular capillary
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Granular casts
Granular casts can result either from the breakdown of cellular casts or the inclusion of aggregates of plasma proteins (e.g., albumin) or immunoglobulin light chains
indicative of chronic renal disease
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Granular cast
Non-specific but usually pathologic
Usually the ‘granules’ are from the degeneration of cellular casts
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Waxy casts
waxy casts suggest severe, longstanding kidney disease such as renal failure(end stage renal disease).
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Waxy casts
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Waxy cast
Thought to result from the degeneration of cellular casts
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Fatty casts Formed by the breakdown of lipid-rich
epithelial cells, these are hyaline casts with fat globule inclusions
They can be present in various disorders, including
nephrotic syndrome, diabetic or lupus nephropathy, Acute tubular necrosis
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Fatty casts
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Pigment casts
Formed by the adhesion of metabolic breakdown products or drug pigments
Pigments include those produced endogenously, such as
hemoglobin in hemolytic anemia, myoglobin in rhabdomyolysis, and bilirubin in liver disease.
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Crystal casts
Though crystallized urinary solutes, such as oxalates, urates, or sulfonamides, may become enmeshed within a hyaline cast during its formation.
The clinical significance of this occurrence is not felt to be great.
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Red cell casts
The presence of red blood cells within the cast is always pathologic, and is strongly indicative of glomerular damage.
They are usually associated with nephritic syndromes.
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Erythrocyte cast
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White blood cell casts
Indicative of inflammation or infection, pyelonephritis acute allergic interstitial nephritis, nephrotic syndrome, or post-streptococcal acute
glomerulonephritis
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Leucocyte cast
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White blood cell cast
Seen in interstitial disease most commonly
Also seen in glomerulonephritis
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WBC Cast Urine:
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Mixed Cast Urine:
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Epithelial casts
This cast is formed by inclusion or adhesion of desquamated epithelial cells of the tubule lining.
These can be seen in acute tubular necrosis and toxic ingestion, such as from mercury,
diethylene glycol, or salicylate.
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Non-renal epithelial cells
Uro-epithelium is columnar If there are a lot of these
it means there is sloughing of the system
Skin is squamous If there are a lot of
squamous it is a contaminated specimen
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Hyaline cast
Hyaline casts are difficult to see due to their low refractive index. This one is granular, which makes it easier to visualize. (Bright field microscopy, 200X magnification.)
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Red blood cell cast
This slide shows a red blood cell cast with red blood cells concentrated at one end.(Bright field microscopy, 160X magnification)
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White blood cell cast
This is a white blood cell cast with intact leukocytes. The leukocytes have multilobed nuclei. (Bright field microscopy)
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Coarsely granular cast
This coarsely granular cast is easily seen with interference-contrast microscopy at 160X magnification.
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Waxy cast
This is a waxy cast seen in urine with interference-contrast microscopy at 160X magnification. The cast is surrounded by red blood cells and segmented neutrophils.
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So…what is a cast???
Protein based cylindrical molds of the renal tubule
Form in distal tubules and collecting ducts Result of damage to the renal tubule Can contain cells and other material Dehydration and acidic urine especially
predisposes to cast formation
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Formation of Casts:
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Red cell cast
Almost always associated with glomerulonephritis or vasculitis
Virtually exclude extra-renal causes of bleeding
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Red cell Casts in Urine:
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Epithelial Casts in Urine:
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Granular Casts in Urine:
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Crystals in urine
Crystals in acidic urine
Uric acid Calcium oxalate Cystine Leucine
Crystals in alkaline urine
Ammonium magnesium phosphates(triple phosphate crystals)
Calcium carbonate
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crystals
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Urine Oxalate Crystals:
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Calcium oxalate crystals in urine
Calcium oxalate crystals are usually found in acid urine. They commonly appear octahedral.(Brightfield microscopy, 100X magnification.)
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Amorphous phosphates in urine
Amorphous phosphates appear in neutral to alkaline urine as fine, colorless or slightly brown granules. White precipitate is observed on centrifugation. (Brightfield microscopy, 160X magnification.)
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Triple phosphate crystals in urine
Triple phosphate crystals, viewed here with interference-contrast microscopy (160X magnification), resemble prisms or "coffin lids". They are found normally in alkaline or neutral urine. They are colorless.
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Ammonium biurate crystals in urine
Ammonium urate crystals are easily distinguished by their golden brown color and "thorn apple" shape. They are seen here under bright field microscopy (160X magnification). They are the only urate crystals that appear in alkaline urine.
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Calcium phosphate crystals in urine
Calcium phosphate crystals assume various forms including the rosette and pointed finger forms shown here with bright field microscopy (160X magnification). They appear most often in alkaline urine.
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Amorphous urates in urine
Amorphous urates appear as fine pink or brownish-tan granules (brightfield microscopy, 200X magnification). They are salts of uric acid and are normally found in acid or neutral urine.
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Uric acid crystals in urine
Uric acid crystals exhibit extreme pleomorphism in size and in shape. They appear readily in acid urine allowed to stand at room temperature. (Bright field microscopy, 160X magnification)
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Calcium carbonate crystals in urine
Calcium carbonate crystals are small and colorless and appear in alkaline urine as granules or as small dumbbells. Bacteria are also present in this field (brightfield microscopy, 250X magnification).
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Cystine crystals in urine
Cystine, an amino acid, is an abnormal finding in urine. Rarely seen, these crystals are found in acid urine and are seen as thin, colorless, hexagonal plates. (Brightfield microscopy, 160X magnification.)
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Tyrosine crystals in urine
Tyrosine crystals are not normally found in urine. They are products of protein metabolism and appear in urine of people with tissue degeneration or necrosis (acute liver disease, severe leukemia, typhoid fever, and smallpox). They are present only when urine is acid. They are colorless to yellowish brown, needle shaped crystals and have a fine silky appearance. The needles may be single or arranged in sheaves or rosettes. Tyrosine crystals usually appear in urinary sediment together with leucine crystals
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Leucine crystals in urine
Leucine crystals are not normally found in urine They appear in urine in association with tyrosine and are manifestations of the same clinical conditions. When found, leucine crystals are in acid urine in the form of spheroids with concentric striations. They are dense, highly refractive and appear as yellowish brown bodies. (Brightfield, 160X magnification)
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Cholesterol crystals in urine
Cholesterol crystals are seen in the center of this field with squamous epithelial cells on either side. Cholesterol crystals are found in acid or neutral urine. They appear as regular or irregular transparent plates. They may occur singly or in large numbers. Usually one or more corners are cut off or notched, justifying their description as "stair step crystals". They are not commonly seen and are always considered pathological. They can be found in various renal diseases. (Brightfield microscopy, 160X magnification.)
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Sulfa crystals in urine
Sulfonamide crystals form primarily in acid urine. The shape and color of these crystals are extremely variable, depending on the particular sulfonamide being administered to the patient. The most common forms encountered include rosettes, fan shapes and those resembling shocks of wheat. Sulfa crystals have pathologic significance, since they tend to form renal calculi that may damage renal tubules. (Brightfield microscopy, 160X magnification.)
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Bacteria and white blood cells
This is a low power view (100X magnification) of urine with bacteria and several leukocytes in the field. Bacteria are bacillary and diffusely scattered throughout. The urine is stained with Sternheimer-Malbin stain.
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Budding yeast cells
Budding yeast are visible on the left side of this slide with a squamous epithelial cell on the right. There are no segmented neutrophils seen. This suggests the yeast are a contaminant and not causing a urinary tract infection (bright field microscopy, 200X).