urine analysis dr. ahmadvand introduction urine is formed in the kidneys, is a product of...

Urine analysis

Dr. Ahmadvand

Introduction Urine is formed in the kidneys, is a product

of ultrafiltration of plasma by the renal glomeruli.

Formation of Urine

Three processes of urine formation: glomerular filtration tubular reabsorption tubular secretion

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

Collection of urine

Early morning sample-qualitative Random sample- routine 24hrs sample- quantitative Midstream sample

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

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

Urinalysis

Evaluation of urine to obtain information about body health and disease

Three types of testing: Physical Chemical Microscopic

Urine examination

Macroscopic examination Chemical examination Microscopic examination

Macroscopic examination

Volume Color Odour Urinary pH Specific gravity

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

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

Urinary volume

Normal = 600-1550ml Polyuria- >2000ml Oliguria-<400ml Anuria-complete cessation of

urine(<200ml)

Causes of polyuria

Diabetes mellitus Diabetes insipidus Diuretics Intravenous saline/glucose

oliguria

Dehydration-vomiting, diarrhoea, excessive sweating

Renal ischemia Acute tubular necrosis Obstruction to the urinary tract

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

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

Acidic urine

Ketosis-diabetes, starvation, fever Acidosis Acidification therapy

Alkaline urine

Strict vegetarian Systemic alkalosis Alkalization therapy

Odour

Normal= aromatic due to the volatile fatty acids

Ammonical – bacterial action Fruity- ketonuria

Specific gravity

Depends on the concentration of various solutes in the urine.

Measured by-

- refractometer

- dipsticks

High specific gravity

Normal-1.016-1.022 Causes

All causes of oliguria

Gycosuria

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

Apply Your Knowledge

What is the specific gravity shown on this refractometer screen?

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)

BioMedica Diagnostics Inc. / D. Jette / March 2003

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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)

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

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

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.

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%)

Benedict’s test

Detects all reducing substances like glucose, fructose, & other reducing sustances.

To confirm it is glucose, dipsticks can be used (glucose oxidase)

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.

Ketone bodies

3 types Acetone Acetoacetic acid β-hydroxy butyric acid

They are products of fat metabolism

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

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.

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)

Other tests

Sulphosalicylic acid test Dipsticks Esbach’s albuminometer- for quantitative

estimation of proteins

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

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

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

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.

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.

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

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.

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

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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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

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.

The main advantage of dipsticks is that they are

1. convenient,

2. easy to interpret,

3. and cost-effective

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).

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

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

Contents of normal urine m/s

Contains few epithelial cells, occasional RBC’s, few crystals.

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.

Red blood cells in urine

These are red blood cells found in urinary sediment under bright field microscopy (160x magnification).

WBC in Urine :

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.

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.

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).

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)

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.

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.

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

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

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

Granular cast

Non-specific but usually pathologic

Usually the ‘granules’ are from the degeneration of cellular casts

Waxy casts

waxy casts suggest severe, longstanding kidney disease such as renal failure(end stage renal disease).

Waxy casts

Waxy cast

Thought to result from the degeneration of cellular casts

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

Fatty casts

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.

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.

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.

Erythrocyte cast

White blood cell casts

Indicative of inflammation or infection, pyelonephritis acute allergic interstitial nephritis, nephrotic syndrome, or post-streptococcal acute

glomerulonephritis

Leucocyte cast

White blood cell cast

Seen in interstitial disease most commonly

Also seen in glomerulonephritis

WBC Cast Urine:

Mixed Cast Urine:

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.

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

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.)

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)

White blood cell cast

This is a white blood cell cast with intact leukocytes. The leukocytes have multilobed nuclei. (Bright field microscopy)

Coarsely granular cast

This coarsely granular cast is easily seen with interference-contrast microscopy at 160X magnification.

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.

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

Formation of Casts:

Red cell cast

Almost always associated with glomerulonephritis or vasculitis

Virtually exclude extra-renal causes of bleeding

Red cell Casts in Urine:

Epithelial Casts in Urine:

Granular Casts in Urine:

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

crystals

Urine Oxalate Crystals:

Calcium oxalate crystals in urine

Calcium oxalate crystals are usually found in acid urine. They commonly appear octahedral.(Brightfield microscopy, 100X magnification.)

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.)

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.

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.

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.

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.

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)

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).

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.)

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

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)

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.)

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.)

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.

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).

urine analysis dr. ahmadvand introduction urine is formed in the kidneys, is a product of...

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