MICROSCOPIC EXAMINATION OF URINE

SPECIMEN COLLECTION
First morning voiding (contains the most concentrated urine). the morning sample is considered best as it has higher specific gravity and lower ph and is desirable for preservation of formed element. Always record collection time. Use clean, dry and wide mouth container.

Is ideal to analyse the urine within 1 hour of collection.

Purpose of urine analysis

Urinalyses are performed for several reasons: -General evaluation of health -Diagnosis of metabolic or systemic diseases that affect kidney function -Diagnosis of endocrine disorders. For these tests are more usefull the 24 hours urine studies. -Diagnosis of diseases or disorders of the kidneys or uriary tract -Monitoring patients with diabetes

-Testing for pregnancy (by detecting the presence of -HCG hormone in urine)
-Screening for drug abuse

Normal urine

Volume men 1200-1500 ml/24h; women 8001200 ml/24 h

Specific gravity- 1.003-1.020

Ph- acidic (4.7-7.5), average Ph 6.0

Total solids 30-70 g/l

Urine constituents
Anorganic constituents of 24h urine are: - Iron 0.06-0.1 mg - Chloride 9-16 gm - Sodium 3-4 gm

- Phosphate 1.5-2.0 g
- Sulfur 0.7-3.5 gm - Calcium 0.1-0.3 g

Urine constituents
Organic constituents in 24h urine - Nitrogenous compound 35-35 gm

- Urea 15-30 gm
- Creatine 60-150 mg - Ammonia 0.3- 1.0 g - Uric acid 0.3-1.0 g - Protein 0- 0.1 gm

Preservation
All the specimen for routine urinalysis should be examined while fresh (within 1 h of collection). When urine is kept for longer than 1 h before analysis, to avoid deterioration of chemical and cellular material and to prevent multiplication of bacteria, it shoul be stored at 2-8C in a refrigerator.

Cont
The expected changes in the composition of urine stored at room temperature are as follow:
- lysis of RBCs by hypotonic urine - decomposition of casts

- bacterial multiplication
- decrease in glucose level, due to bacterial growth - formation of ammonia from urea by the action of bacteria

Cont
Recommended preservatoves are as follow:
- Toluene

- Formalin - Thymol - Chloroform - Commercial preservatives tabletes. These release formaldehyde

Types of analysis
- Physical examination - Chemical analysis (urine dipstick or by manual methods) - Microscopic examination - Culture - Cytological examination

Physical analysis
Includes : - volume

- color
- appearance - odour

Physical analysis
VOLUME- for adults normal average daily volume of urine is about 1200-1500 ml, the normal range of 24 hr. urine may be from 600-2000 ml.

COLOR the color of normal urine may vary from pale yellow to dark amber. Very pale or colorless urine can result from high fluid consumption, diuretic drugs, natural diuretic such as alcohol and coffee and also in clinical condition such as diabetes insipidus and diabetes mellitus

Physical analysis
APPEAREANCE- normal urine is usually clear. Urine may appear cloudyor turbid from the presence of leukocytes and epithelial cells. This can be confirmed by microscpic examination. Bacteria can also cause cloudiness to urine. Fat can give urine a milky colour. Presence of RBCs may give urine turbid and smoky appearance.
ODOUR presence of ketone bodies gives urine a fruity smell ( green apples). A contaminated urine with bacteria may give a pungent smell due to formation of ammonia

Chemical analysis
The routine urinalysis includes chemical testing for: - protein - glucose - ketone bodies - occult blood - bile salts - urobilinogen

FORMATION OF URINE
URINE is formed through a combination of basic processes: 1) 2) 3) 4) Glomerular filtration Tubular reabsorbtion Tubular secretion and Water conservation

Blood is under high pressure in the glomerulus: thus, plasma (except plasma proteins) moves into the glomerular capsule. This is reffered to as a tubular fluid. Most of the water and many other molecules are reabsorbed into the blood, while some substances are secreted into the tubular fluid. Once the fluid moves into the collecting duct it is called urine. While in the collecting duct , additional water is removed from the urine, concentrating the wastes.

Microscopic examination of the urine

The microscopic examination is a valuable diagnostic tool for the detection and evaluation of renal and urinary tract disorders and other systemic diseases.
The microscopic elements present in the urine (in suspension) are collected in the form pf deposit by centrifugation. A small drop of sediment is examined by making a coverslip preparation under microscope

Procedure
Mix the urine and por into a centrifuge tube (or small test tube) untill is fill (5 ml).
Centrifuge with another balanced test tube for 5 min at 2500 rpm. Pour off the supernatant quickly and complete into another test tube. This can be used for protein determination. Resuspend the deposits by shaking the tube. Place one drop of the deposits on a glass slide. Cover it with coverslip and mark it with identificaton number. Observe it first under low power objective in partially closing the diaphragm and the adjusting the condenser downward untill satisfactory contrast is obtained. Note the content if various fields. Switch to high dry objective and observe at least 10 to 15 different fields.

Requirements
- centrifuge tube or test tube (10*15 mm) - glass slide - cover slips - Pasteur pipettes

- centrifuge
- Microscope - freshly voided urine is required

Observations
Organised elements: RBSs, WBCs, epithelial cells, casts (hyaline, cellular, granular, waxy, fatty). Unorganised elements in acidic urine: calcium oxalate, uric acid, amorphous urate, sodium urate, calcium sulphate, cystine crystals, tyrosine, leucine.

Cont
In alcaline urine: triple phosphate, amorphous phosphate, calcium carbonate, calclium phosphate, ammonium biurates

Artifacts
- Starch crystals - Fibers - Oil droplets - Hair

- Air bubbles

Organised elements
ERYTHROCYTES
In fresh urine these cells have a normal , pale yellow appearance. They appear smooth, biconcave disks about 7m in diameter and 2 m thick. Thet do not contain nuclei. In hypotonic urine the red cells swell up and lyse. In hypertonic urine the red cells crenate.

Microscopic Examination

LEUCOCYTES The puss cells can enter in urine anywhere from the glomerulus to the urethra. Normal urine can contain 2-3 pus cells/ per h.p.f.

There are mostly neutrophils.

Approximate diameter: 10-12 m.

Microscopic Examination
WBCs

Epithelial cells
Originate from any site in the genitourinary tract from the proximal convoluted tubule to the urethra or from vagina.

Normally few cells (3-5) per h.p.f from these sites can be found in the urin.
Three types of epithelial cells may be recognized - Tubular. - Transitional - Squamous

Tubular epithelial
These are slightly larger then leucocytes and contain large round nucleus.

They may be cuboidal, flat or columnar.

Microscopic Examination
Tubular Epithelial Cells

Transitional epithelial
These are two to four times as large as white cells.
They may be pear shaped or round. Occasionally these cells may contain two nuclei.

Microscopic Examination
Transitional Cells

Squamous epithelial
These are large, flat and irregular in shape. They contain abundant cytoplasm and small central nuclei.

Microscopic Examination
Squamous Cells

Casts
Urinary castes are formed in the lumen of the tubule of the kidney. The renal tubules secrete a mucoprotein called Tamm-Horsfall protein which is believed to form the basis matrix of all casts. Casts dissolve in alkaline urine. Castes have nearly parallel sides and rounded or blunted ends. These are the following tpes: - Granular - Hyaline - Red cell casts - Epithelial cell casts - Waxy casts - Fatty casts

Granular casts
These always indicate significant renal disease. The casts are present due to direct aggregation of serum protein in a Tamm-Horsfall mucoprotein matrix.

Microscopic Examination
Granular casts

Hyaline casts
They are colorless, homogeneous, transparent and with rounded ends.

These casts can be seen in increased number even in the mildest kind of renal disease.
A few hyaline casts may be present in normal urine.

Microscopic Examination
Hyaline casts

Red cell casts

The cast may contain only a few RBCs in protein matrix or may be many cells packed close together with no visible matrix. Presence of red cell is always pathogenic. They are usually diagnostic of glomerular disease being found in acute glomerulonephritis and also in renal infarction.

Microscopic Examination
RBCs casts

White cell casts

The majority of white cells that appear in casts are polymorphonuclear neutrophils.

The cells may be few or many, tightly packed together.

The casts may be present in renal infarction and in glomerular disease.

Microscopic Examination
WBCs Casts

Epithelial cell casts

The epithelial cell may be arranged haphazardly and vary in size and shape. These casts are rarely seen in urine. Presence of these casts degeneration and necrosis. indicate tubular

Present in severe chronic renal disease.

Microscopic Examination
Epith. Casts

Waxy casts
Have high refractive index. These are yellow grey or colorless and have a homogenous appearance. These result from the degeneration of granular casts.

Microscopic Examination
Waxy Casts

Fatty casts
Fatty casts are formed by incorporated free fat droplets or oval fat bodies.

These are frequently seen in nephrotic syndrome and toxic renal poisoning.

Microscopic Examination
Fatty Casts

Unorganized elements
CRYSTALS Crystals present in acidic urine. Uric acid crystals These can occur in most characteristic diamond rhombic or rosette form. These are usually stained with urinary pigments as yellow or red brown. These are soluble in sodium hydroxide and insoluble in hcl. Pathologic conditions: Gout, chronic nephritis, acute febrile condition.

Microscopic Examination
Uric acid crystals in urine

Calcium oxalate crystals

These are colorless and envelop shaped. They also appear as oval, sphere or biconcave disk which have a dumb bell shape (when viewed from side). These are found in acid or neutral urine. These can be present in urine after the ingestion of tomatoes, oranges and vitamin C. Pathological condition: They can be present in diabetes mellitus, liver disease and chronic liver disease.

Microscopic Examination
Calcium oxalate crystals in urine

Calcium oxalate crystals are usually found in acid urine. They commonly appear octahedral.

Amorphous urates
These are urate salts of sodium, potassium, magnesium and calcium.

Usual form is non crystalline and amorphous and appearance is yellow-red granular.
These are soluble in alkali at 60 c.

They have no clinical significance.

Microscopic Examination
Amorphous urates in urine

- appear as fine pink or brownish-tan granules - they are salts of uric acid and are normally found in acid or neutral urine

Sodium urate
-May be present as amorphous or as crystals
-These crustals are colorless or ellowish needles occurring at 60 c -They have no clinical significance

Microscopic Examination
Sodium urate crystals in urine

Calcium sulfate crystals

-

These are long, thin and colorless needles or prisms

-These are soluble in acetic acid

-These are rarely seen in the urine and they have no clinical significance

Microscopic Examination
Calcium sulfate crystals in urine

Hippuric acid crystals

These

are in the form of elongated prisms or plates

The color may be yellow brown or colorless These are soluble in water

Are rarely seen in urine and have no clinical significance

Microscopic Examination
Hippuric acid crystals in urine

Cystine crystals
- These are colorless, hexagonal plates with equal or unequal sides

- Are soluble in hydrochloric acid and ammonia

- Pathological condition : they occur in patients with either congenital cystinosis or congenital cystinuria; they can form calculi

Microscopic Examination
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

Tyrosine
- Appear in the form of fine, refractile needles, occurring in clusters

- These are soluble in ammonium hydroxide

- Pathological conditions: severe liver disease and tyrosinosis

Microscopic Examination
Tyrosine crystals in urine

Tyrosine crystals
- Tyrosine crystals in urine are not normally found in urine - They are producs of protein metabolism and appear in ruine 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, neddle shaped crystals and have a fine silky appearance. - The needles may be single or arranged in sheaves or rosettes - Ussualy appear in urinary sediment together with leucine crystals

Leucine
- These crystals are oily, highly refractile sheroids - They have yellow or brown color - These crystals are soluble in hot acohol and acetic acid - Pathological conditions-severe hepatitis, acute yellow atrophy

Microscopic Examination
Leucine crystals in urine

Leucine crystals
- Are not normaly found in urine - They apperar 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 sheroids with concentric striatons. These are dense, highly refractive and appear as yellowish brown bodies

Cholesterol crystals
- Are large, flat and in the form of transparent plates with notched corners

- They are soluble in either chloroform and hot alcohol

- Pathological conditions: nephritis, contions, excessive tissues breakdown nephrotic

Microscopic Examination
Cholesterol crystals

Sulfa crystals
- Most of the sulfa drugs precipitate out as sheaves of needles

- They may be clear or brown in color and usually appear with eccentric binding
- These drugs are soluble in acetone

Microscopic Examination
Sulfa crystals in urine

Sulfa crystals
-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 shaped and those resembling shocks of wheat -Sulfa cystals have pathologic significance, since they tend to form renal calculi that may damage renal tubules

Triple phosphates
- Crystals found in alkaline urine - The crystals are colorless prisms with three to six sides and frequently with oblique end - Is soluble in acetic acid - Frequently found in normal urine - Pathological conditions: chronic cystitis, chronic pyelitis, enlarged postate

Microscopic Examination
Triple phosphate crystals in urine

- Resemble prisms or coffin lids

- They are found normally in alkaline or neutral urine

- They are colorless

Amorphous phosphates
- These are present in amorphous granular form - They have no clinical significance

Microscopic Examination
Amorphous phosphates in urine
- Appear in neutral to alkaline urine as fine, colorless or slightly brown granules - White precipitate is observed on centrifugation

Calcium carbonate
- Appear as small, colorless and in the form of spherical, dumbbell shape or as granular type

- They have no clinical significance

Microscopic Examination
Calcium carbonate crystals in urine

- Are small crystals and colorless - Appear in alkaline urine as granules of as small dumbbelss - Bacteria are also present in this field

Calcium phosphate
- There are long, thin and colorless - The appearance is like prisms with one pointed end, arranged as rosettes or stars - Soluble in dilute acetic acid - May be present in normal urine and have no clinical significance

Microscopic Examination
Calcium phosphate crystals in urine
- They assume various forms including the rosette and pointed finger forms
- They appear most often in alkaline urine

Ammonium biurates
- These are yellow brown or sherical bodies with or without long, irregular spicules - Presence in abnormal if they are dound in fresh urine

Microscopic Examination
Ammoium biurate crystals in urine

- Are easily distiniguished by their golden brown color and thorn apple shape. - They are the only urate crystals that appear in alkaline urine

Artifacts
- Starch crystals : are found in urine as round or oval and highly refractile crystals

- Fibers : they may come from clothing, toilet paper or may be lint from the air
- Oil droplets : are present as the result of contamination from lubricants. They are sherical and vary in size - Hair - Air bubbles - Parasites

Artifacts

Artifacts