PATHOLOGICAL OR ABNORMAL URINE

CONSTITUENTS
:I- Proteinuria

- Proteinuria is a condition in which urine contains an abnormal amount of protein.

- The majority of proteins found in the urine arise from the blood.

- As blood passes through healthy kidneys, they filter the waste products out and

leave in the things the body needs, like proteins. Most proteins are too big to pass

through the kidneys' filters into the urine unless the kidneys are damaged. The main

protein that is most likely to appear in urine is albumin. Proteins from the blood can

escape into the urine when the filters of the kidney, called glomeruli, are damaged.

Sometimes the term albuminuria is used when a urine test detects albumin

specifically.

- Large amounts of protein in the urine may cause it to look foamy. Also, because the

protein has left the body, the blood can no longer soak up enough fluid leading to

swelling in the hands, feet, abdomen, or face. These are signs of very large protein

loss.

- In a healthy renal and urinary tract system, the urine contains no protein or only

traces amounts.

- Because albumin is filtered more readily than the globulins, it is usually abundant in

pathological conditions. Therefore, the term albuminuria is ~~~= proteinuria.

- The qualitative tests for proteinuria are most commonly performed using a reagent

test strip. These methods depend on the change in the response of an indicator dye in

the presence of protein. (See the figure)

- Quantitative examination of urine protein requires considerable attention to

the volume or time of urine collection because the concentration may vary with

time and volume.

- Most assays are performed on urine sample of 12-24h.

- Reference Value (RV) for 24-h urine Male = 1-4 mg/dl

Female = 3-10mg/dl

Child = 1-10mg/dl

Reference value – Qualitative Normal = Negative -

Qualitative Tests for Proteins

.Before performing a test for albumin, the urine should be filtered or centrifuged -
:Heat Coagulation Test -1
Fill a test tube one third full of urine and gently heat the upper half of the fluid to

boiling, being careful that this fluid does not mix with the lower half which serves as

a control. Turbidity indicates proteins or calcium or magnesium phosphate. Acidify

the urine slightly by the addition of 3-5 drops of dilute acetic acid where upon the

turbidity if due to phosphate will disappear.

:Heller's Test -2

.Place 3ml of conc. HNO3 in test tube -1

Tie the tube to about a 30 degree angle. Using a pipette allow the urine to flow -2

.slowly down the side of the tube

If albumin is present, within a few minutes a white ring will form at the -3

.junction between the two liquids

:II- Glucosuria

.Glucosuria is a condition in which urine contains an abnormal amount of glucose -

Glucose is present in glomerular filtrate and is reabsorbed by the proximal tubule. -

- If the blood glucose level exceeds the reabsorption capacity of the tubules, glucose

.will appear in the urine

Tubular reabsorption of glucose is by active transport in response to the body's -

.need to maintain an adequate concentration of glucose

The blood level at which tubular reabsorption stops is termed renal threshold -

.which for glucose is between 160-180 mg/ dl

Glucosuria occurs in diabetes mellitus, which characterized by abnormal blood-

glucose level (hyperglycemia) and usually increased volume of urine. The urine may

.be light in color and have a high specific gravity

Qualitative Tests for Glucose

Before testing for sugar the urine should be free of albumin. If present, it must be -

removed by heat coagulation. Firstly, acidify the urine with dilute acetic acid, boil,

.cool and filter

:Principles of Fehling' and Benedict's reagent

I- Fehling's Test

In this test the presence of aldehydes but not ketones is detected by reduction of -
the deep blue solution of copper (II) to a red precipitate of insoluble copper oxide.
The test is commonly used for reducing sugars but is known to be NOT specific for
aldehydes. For example, fructose gives a positive test with Fehling's solution as does
.acetone

:Two solutions are required

Fehling's "A" uses 7 g CuSO4.5H2O dissolved in distilled water containing 2 drops -

.of dilute sulfuric acid
Fehling's "B" uses 35g of potassium tartrate and 12g of NaOH in 100 ml of -
.distilled water

.These two solutions should be stoppered and stored until needed -

:Method
"Mix 1ml of solution-"A" with 1ml of solution-"B-

.Add 1 ml of this mixture to an empty test tube -

Add 1ml of the urine in test tube. If the sample is from patient the sample must -
.first boil l to liberate ammonia which may have a destructive effect on the reagent

.Mix urine with Fehling's reagent -

.Place the tube in a water-bath at 60°C -
.A positive test is indicated by a green suspension and a red precipitate -

The test is sensitive enough that even 1 mg of glucose will produce the -
.characteristic red colour of the compound

Copper-tartrate-complex

II- Benedict's reagent

- Also called Benedict's solution or Benedict's test is a reagent used as a test for the

presence of reducing sugars (such as glucose, lactose, and fructose, but not sucrose),

or more generally for the presence of aldehydes, in a solution.

- Benedict's reagent contains blue copper(II) sulfate (CuSO4) · 5H2O which is

reduced to red copper(I) oxide by aldehydes, thus oxidizing the aldehydes to

carboxylic acids.
- The copper oxide is insoluble in water and so precipitates. The colour of the final

solution ranges from green to brick red depending on how many of the copper (II)

ions are present.

- Benedict's reagent can be used to test for the presence of glucose in urine. Glucose

found to be present in urine is an indication of diabetes. 5.0ml of Benedict's

qualitative solution is mixed with 0.5ml of urine and the mixture is put in a boiling

water bath for 5 minutes.

- The results are recorded thus:

No precipitate -

Green a trace

Yellow +

Orange ++

Red +++

- Once sugar is detected in urine, further tests have to be undergone in order to

determine which sugar is present. Only glucose is indicative of diabetes.

- Benedict's quantitative reagent is used to determine how much sugar is present.

- This solution forms as white precipitate rather than a red one because the amount

of Cu reduced is obtained by complexing with KSCN giving CuSCN which is white

ppt.

- The disappearance of blue color indicates the complete reduction of the Cu

present.

- Potassium ferrocyanide is added in order to prevent the reoxidation of the Cu.

Redox reaction

Method:

-1ml Benedict + 1ml urine ====>> BWB (5min.) ===>>>Red ppt

NV – random sample -Qualitative Normal = Negative -

NV- 24-h urine- Quantitative 1-15mg/dl -

 Glucometer

:III-Ketonuria

Ketonuria is a condition in which urine contains an abnormal amount of ketone -

.bodies

- Ketone bodies are three chemicals that are produced as by-products when fatty
acids are broken down for energy. Any production of ketone bodies is called
ketogenesis, and this is necessary in small amounts. But, when excess ketone bodies
accumulate, this abnormal (but not necessarily harmful) state is called ketosis.
When even larger amounts of ketone bodies accumulate such that the body's pH is
lowered to dangerously acidic levels, this state is called ketoacidosis.

- The three ketone bodies are acetoacetate, beta-hydroxybutyrate and acetone. The
first two are not technically ketones. (They are called ketone bodies because they
come from ketones).

- The exhalation of acetone is responsible for the characteristic "fruity" odor of the
breath of persons in ketosis states.

- Both acetoacetate and beta-hydroxybutyrate are acidic, and, if levels of these

ketone bodies are too high, the pH of the blood drops, resulting in ketoacidosis. This
happens in untreated Type I diabetes and subsequent starvation.

- Urine ketones measurement frequently provides a more reliable indicator of

acidosis than blood testing because they appear in the urine before there is any
significant increase in the blood.
- How to prepare for the test?

A special diet may be recommended, and you should stop taking any drugs that may
affect the test.

If the collection is being taken from an infant, extra collection bags may be
necessary.

Why the test is performed ?

Ketones (beta-hydroxybutyric acid, acetoacetic acid, and acetone) are the end-
product of rapid or excessive fatty-acid breakdown. As is the case with glucose,
ketones will be present in the urine when the blood levels of ketone surpass a certain
threshold.

Fatty-acid release from adipose tissue (body fat) is stimulated by a number of

hormones including glucagon, epinephrine, and growth hormone. The levels of these
hormones increase in starvation (whether related to excess alcohol use or not),
uncontrolled diabetes mellitus, and a number of other conditions.

Normal Values

A negative test result is normal. Results of the presence of acetone in the urine are
usually listed as small, moderate or large with these corresponding values:

Small - < 20 mg/dL

Moderate - 30-40 mg/dL

Large - > 80 mg/dL

What abnormal results mean ?

A positive test may indicate:

 • Metabolic abnormalities, including uncontrolled diabetes or glycogen
storage disease
• Abnormal nutritional conditions, including starvation, fasting,
anorexia, high protein or low carbohydrate diets
• Vomiting frequently over a long period of time, including hyperemesis
gravidarum (a severe form of morning sickness)
• Disorders of increased metabolism, including hyperthyroidism, fever,
acute or severe illness, burns, pregnancy, lactation (nursing a baby), or post-
surgical condition

Special considerations

Special diets may alter test results. For example, a diet consisting of low amounts of
carbohydrates with high amounts of protein and fat may affect the ketone levels.

Drugs that may cause false positive measurements include glucocorticoids.

- In healthy persons, ketones are formed in liver and completely metabolized so that

only negligible amounts appear in the urine.

- During pregnancy, the early detection of ketones is essential because ketoacidosis

is a prominent factor that contributes to intrauterine death.

Qualitative tests for Ketone Bodies

1ml sat. (NH3)2SO4 + 2ml urine + 1/2ml freshly prepared sodium nitroprosside + -

conc. ammonia (1-2ml) =====>>>>mix ===>>>> Deep permanganate color after

.few minutes indicate the presence of acetone

NV – random sample -Qualitative Normal = Negative -

:IV- Hematuria and Hemoglobinuria

- Hematuria is the presence of red blood cells (RBCs) in the urine. In microscopic

hematuria, the urine appears normal to the naked eye, but examination under a

microscope shows a high number of RBCs (see the figure). Gross hematuria can be

seen with the naked eye—the urine is red or the color of cola.

- Several conditions can cause hematuria, most of them not serious. For example,

exercise may cause hematuria that goes away in 24 hours. Many people have

hematuria without any other related problems. Often no specific cause can be
found. But because hematuria may be the result of a tumor or other serious

problem, a doctor should be consulted.

To find the cause of hematuria, or to rule out certain causes, the doctor may order a

series of tests, including urinalysis, blood tests or others.

- Urinalysis is the examination of urine for various cells and chemicals. In addition

to finding RBCs, the doctor may find white blood cells that signal a urinary tract

infection or casts (groups of cells molded together in the shape of the kidneys' tiny

filtering tubes) that signal kidney disease. Excessive protein in the urine also signals

kidney disease.

- Blood tests may reveal kidney disease if the blood contains high levels of wastes

that the kidneys are supposed to remove.

Hemoglobinuria: results from hemolysis i.e. the rupturing of erythrocyte and the
liberation of hemoglobin. This may occur in malaria, typhoid, yellow fever,
hemolytic jaundice and other diseases.

Benzidine reaction:

- This reaction depends on the Fe ion found in hemoglobin.

Method:

- 0.5ml urine + 1.5ml benzidine, then add 0.5ml H2O2 >>>>>> Blue color.

V- Bilirubin (Bile):

- Bilirubin is the waste product that results from the breakdown of hemoglobin
molecules from worn out red blood cells. Ordinarily, it is excreted from the body as
the chief component of bile. Excessive levels of bilirubin stain the fatty tissues in the
skin yellow; this condition is called jaundice.

- Bilirubin is found in the dark urine of obstructive jaundice and later in the course
of hepatitis. Urobilinogen is found in early hepatitis. It is colorless but darkens on
standing. Bile pigments are included in the routine strip used in urinalysis. Positive
results are an indication for measuring s. bilirubin and liver enzymes.

Test for bile pigments:

Method:

- 1ml urine + 5 drops 1% iodine in alcohol are added >>>>>> Green ring indicates
the presence of bile
pigments.

Note: In all clinical labs qualitative urinalysis depends on using dipstick (strip)
to determine the normal and abnormal constituents of urine (see the figure).
 RESULTS

You are supplied with the sample -

.of urine, carry on routine UA
.Detect the presence of any pathological constituents -
?According to what you studied in the lab, is this urine normal or not -
.Present your results in a good and full lab report -
 III- Microscopic Examination

- A sample of well-mixed urine (usually 10-15 ml) is centrifuged in a test tube

at relatively low speed (about 2-3,000 r.p.m) for 5-10 minutes until a moderately

cohesive button is produced at the bottom of the tube.

- The supernatant is decanted and a volume of 0.2 to 0.5 ml is left inside the
tube.
- The sediment is resuspended in the remaining supernatant by flicking the

bottom of the tube several times.

- A drop of resuspended sediment is poured onto a glass slide and cover-

slipped.
 - The sediment is first examined under low power to identify most crystals,

casts, squamous cells, and other large objects. Next, examination is carried out

at high power to identify crystals, cells, and bacteria.

Examination

The sediment is first examined under low power to identify most crystals, casts,
squamous cells, and other large objects. Next, examination is carried out at high
power to identify crystals, cells, and bacteria.

White Blood Cells

Pyuria refers to the presence of abnormal numbers of leukocytes that may appear with
infection in either the upper or lower urinary tract or with acute glomerulonephritis.
Usually, the WBC's are granulocytes. White cells from the vagina, especially in the
presence of vaginal and cervical infections, or the external urethral meatus in men and
women may contaminate the urine.

Epithelial Cells

Renal tubular epithelial cells, usually larger than granulocytes, contain a large round or
oval nucleus and normally slough into the urine in small numbers. However, with
nephrotic syndrome and in conditions leading to tubular degeneration, the number
sloughed is increased.

Casts

Urinary casts are formed only in the distal convoluted tubule (DCT) or the collecting duct
(distal nephron). The proximal convoluted tubule (PCT) and loop of Henle are not
locations for cast formation. Hyaline casts are composed primarily of a mucoprotein
(Tamm-Horsfall protein) secreted by tubule cells. The Tamm-Horsfall protein secretion
(green dots) is illustrated in the diagram below, forming a hyaline cast in the collecting
duct:
Red blood cells may stick together and form red blood cell casts. Such casts are
indicative of glomerulonephritis, with leakage of RBC's from glomeruli, or severe
tubular damage.

White blood cell casts are most typical for acute pyelonephritis, but they may also
be present with glomerulonephritis. Their presence indicates inflammation of the
kidney, because such casts will not form except in the kidney.
Bacteria

Bacteria are common in urine specimens because of the abundant normal microbial
flora of the vagina or external urethra and because of their ability to rapidly
multiply in urine standing at room temperature. Therefore, microbial organisms
found in all but the most scrupulously collected urines should be interpreted in view
of clinical symptoms.

Diagnosis of bacteriuria in a case of suspected urinary tract infection requires

culture.

Yeast

Yeast cells may be contaminants or represent a true yeast infection. They are often
difficult to distinguish from red cells and amorphous crystals but are distinguished
by their tendency to bud. Most often they are Candida, which may colonize bladder,
urethra, or vagina.
Crystals

Common crystals seen even in healthy patients include calcium oxalate, triple
phosphate crystals and amorphous phosphates.

Very uncommon crystals include: cystine crystals in urine of neonates with

congenital cystinuria or severe liver disease, tyrosine crystals with congenital
tyrosinosis or marked liver impairment, or leucine crystals in patients with severe
liver disease or with maple syrup urine disease.

WBC RBC
Epithelia cells Casts

Crystals

RBC CAST
WBC, RBC and bacteria