Clinical Biochemistry

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CLINICAL BIOCHEMISTRY

 The systematic study of biochemical process associated with health

and diseases and the measurement of constituents in body fluids or
tissues to facilitate diagnosis of diseases to asses the clinicians in

 1- confirming or rejecting a diagnosis

 2- providing guidelines in patient management

 3- Establishing a prognosis

 4- detecting a disease through case finding or screening

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 5- monitoring follow up therapy
BIOLOGICAL FLUIDS & URINE ANALYSIS
 BIOLOGICAL FLUIDS
 Biological materials acquired or derived from living or deceased
human beings collected via intervention for research purposes or
part of the standard care including but not limited to the following:
 Amniotic fluid
 Cerebrospinal fluid

 Ascitic fluid

 Plural fluid

 Blood

 Plasma

 Serum

 Synovial Fluid

 Urine

 Semen
ASCITIC FLUID
 Ascitic fluid: the accumulated fluid in the peritoneal cavity.
 A sample of fluid is typically obtained using a needle and syringe
“paracentesis”
 SERUM ASCITIC ALBUMIN GRADIENT (SAAG)
The serum ascitic albumin gradient (SAAG) indirectly measures portal
pressure and can be used to determine if ascites is due to portal
hypertension.

SAAG calculation

SAAG = (serum albumin) – (ascitic fluid albumin)

 Interpretation

A high SAAG (>1.1mg/dL) suggests the ascitic fluid is a transudate.

A low SAAG (<1.1mg/dL) suggests the ascitic fluid is an exudate 6

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TRANSUDATE EXUDATE
A high SAAG (i.e. transudate) Causes of a low SAAG (i.e.
suggests the presence exudate)
of portal hypertension
•Cirrhosis •Malignancy

Congestive heart failure •Infection

Kwashiorkor malnutrition •Pancreatitis

•Fulminant hepatic failure

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 LACTATE DEHYDROGENASE
 Another way of differentiating between an exudate and a transudate

• LDH <225 U/L = transudate

• LDH > 225U/ L = exudate

Biochemistry

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Microscopy

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 PLEURAL FLUID
 Liquid that is located between the layers of the pleura.

 The pleura is a two-layer membrane that covers the lungs and lines the chest
cavity, it keeps the pleura moist and reduces friction between the membranes
during breathing.

 Pleural effusion: too much fluid builds up in the pleural space, prevents the
lungs from fully inflating, making it hard to breathe.

 Thoracentesis: an invasive medical procedure to remove fluid or air from

the pleural space for diagnostic or therapeutic purposes.
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 The presence of food particles suggests oesophageal rupture. 18
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 Biochemistry

Pleural fluid Levels > 1000 IU/L: empyema or malignancy

LDH

Glucose A low glucose level (60 mg/dl) found in empyema, TB,

malignancy.

Cholesterol and TAG > 1.24 mmol/l with a cholesterol level of < 5.18
triglycerides mmol/l is diagnostic of chylothorax.

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WHITE BLOOD CELLS (WBC)

 Transudates have WBC counts less than 1000 cells/µL

 Exudates generally have WBC counts greater than 50,000 cells/µL.

 Pleural fluid lymphocytosis suggests TB or malignancy.

 Neutrophil dominant effusions are associated with empyema or

pulmonary embolism

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 This pleural fluid forms as a filtrate from pleural blood vessels.

 At the same time, it is drained into the lymphatic vessels, and this allows for
regular renewal of the fluid.

 Transudate: (hydrothorax) forms when too much fluid starts to move from
the pulmonary capillaries into the pleural space, either because of increased
hydrostatic pressure or decreased oncotic pressure within the pulmonary
capillaries. Increased hydrostatic pressure occurs in heart failure, where the heart
can’t pump blood effectively, so it backs up into the pulmonary vessels, leading
to pulmonary hypertension; ultimately, the high pressure forces fluid out of the
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pulmonary capillaries and into the pleural space.

 On the other hand, decreased oncotic pressure can be caused
by cirrhosis, which leads to decreased hepatic production of plasma
proteins like albumin; or nephrotic syndrome, where renal filtration of
blood is impaired, so the proteins are lost in urine
 Exudate

 Exudate forms when there’s increased permeability of the pulmonary

capillaries, which allows fluid, immune cells, and large proteins, along
with lactate dehydrogenase or LDH, to leak out of the capillaries and
into the pleural space. This can be caused by trauma, malignancy, such
as lung cancer, inflammatory conditions like pancreatitis, 24

and systemic lupus erythematosus, or an infection like pneumonia.

 Additionally, in some cases, the fluid could be purulent, meaning it
contains pus, which is called an empyema or pyothorax, and is
usually caused by bacterial infection like pneumonia; or the fluid
could be sanguineous, meaning it contains blood, called a
hemothorax, and it’s usually caused by blunt chest trauma, as well as
malignancy, or pulmonary embolism; or it could contain lymph,
which is called a chylothorax, and it can be caused by lymphatic
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system injury from trauma or medical procedures.
DIAGNOSTIC CRITERIA FOR PLEURAL EFFUSION
 Transudate
 Protein <30 g/L (in patients with a normal serum protein level)

 Exudate

 Protein >30 g/L (in patients with a normal serum protein level)

 The fluid is considered an exudate if any of the following are present:

• The ratio of pleural fluid to serum protein is greater than 0.5.

• The ratio of pleural fluid to serum LDH is greater than 0.6.

• The pleural fluid LDH value is greater than two-thirds of the upper limit
of the normal serum value.

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NORMAL PLEURAL FLUID
• Appearance: clear

• pH: 7.60-7.64

• Protein: < 2% (1-2 g/dL)

• White blood cells (WBC): < 1000/mm³

• Glucose: similar to that of plasma

• LDH: <50% plasma concentration

• Amylase: 30-110 U/L

• Triglycerides: <2 mmol/l

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• Cholesterol: 3.5–6.5 mmol/l
 AMNIOTIC FLUID
 The amniotic fluid is the protective liquid contained by the amniotic sac of
a gravid amniote. This fluid serves as a cushion for the growing fetus, but also
serves to facilitate the exchange of nutrients, water, and biochemical products
between mother and fetus.

 Amniocentesis: medical procedure in which a small amount of amniotic fluid,

which contains fetal tissues, is sampled from the amniotic sac surrounding a
developing fetus (usually between weeks 15 and 20 of pregnancy)

 Used in prenatal diagnosis of chromosomal abnormalities and fetal infections

(fetal cellular DNA), birth defects such as Down syndrome, Sickle cell disease, Tay-
Sachs disease, Neural tube defects.
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 SYNOVIAL FLUID
It is a viscous, non-Newtonian fluid found in the cavities of synovial joints. With its
egg white–like consistency.

The principal role of synovial fluid is to reduce friction between the articular
cartilage of synovial joints during movement.

A synovial fluid analysis is a group of tests that

checks for disorders that affect the joints such as

Osteoarthritis, Gout and Rheumatoid arthritis

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CEREBROSPINAL FLUID
 It is an ultrafiltrate of plasma contained within the ventricles of the brain and the
subarachnoid spaces of the cranium and spine. It performs vital functions, including
providing nourishment, waste removal, and protection to the brain

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Urine:
Is an ultra-filtrate of plasma from which glucose, amino acids, water and other substances essential
to body metabolism have been reabsorbed.
Urine carries waste products and excess water out of the body.

Urine consists of:

(96%) (4%)
dissolved solids:
water
(2%) (2%)
Urea Other compounds

Inorganic: Organic:
Cl-, Na, K. creatinine 34
uric acid
trace amounts of:
sulfate, HCO3 etc.)
 URINE ANALYSIS
Why we examine urine?
To diagnose urinary tract infection, liver and kidney dysfunction and determine
carbohydrate metabolism.
Routine Urinalysis (Routine-UA):
 It consists of a group of tests performed as part of physical examination. It involves
macroscopic and microscopic analysis.

Types of analysis: Physical characteristics

 macroscopic analysis:
Chemical analysis
 microscopic examination: urine sediment is examined under microscope to identify
the components of the urinary sediments. 35
 STEPS IN BASIC URINE ANALYSIS
Three steps analysis:
First: physical characteristics of urine are noted and recorded.
Second: urine sediment is examined under microscope to identify the
components of the sediments.
Third: series of chemical tests.

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 TYPES OF URINE SAMPLE
Random sample:
• Sample collected at any time

• It is the most common sample used for routine tests.

Morning sample:
• First urine sample in the morning

• It is the most concentrated sample

• Used for Pregnancy test (for detection of hCG especially within 40 days
of gestation).
Clean catch midstream:
• Genitalia should be cleaned using disinfectant

• First few ml should be discarded & collect the rest in a sterile cup.

• Used in urine culture. 37

 TYPES OF URINE SAMPLE (CONT.)

24 hours urine sample:

• All the urine passed during the day and night and next day 1st sample is
collected.
• Patient asked to urinate and determine the time, all the urine passed during
the day and night is collected and stored in the refrigerator or a
preservative should be used (according to the test).
• At the end of the 24 hrs patient asked to urinate and empty his bladder

• Used for quantitative analysis (A/C).

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 CHANGES OCCUR IN NON PRESERVED SPECIMEN

➢ Transformation of urea to ammonia which increase pH.

urease
➢ Urea 2NH3 + CO2.
➢ (Bacteria)
➢ Increase bacterial number (bacterial growth).
➢ Increase turbidity caused by bacteria & amorphous crystals.
➢ Increase nitrite due to bacterial reduction of nitrate.
➢ Decrease glucose due to glycolysis and bacterial utilization.
➢ Decrease ketones because of volatilization.
➢ Decrease bilirubin from exposure to light. 39

➢ Changes in color due to oxidation or reduction of metabolic sub.

 Direct visual observation.
 Normal fresh urine: Color: pale or dark yellow-amber, clear.

 Vol:750 - 2000 ml/24hr.

 Physical examination involves:

1. Color

2. Transparency

3. Odor

4. Volume

5. pH

6. Specific gravity
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7. Sedimentation
URINE VOLUME:
• Normal: 1- 2.5 L/day
• Oliguria: Urine Output < 400ml/day
• Dehydration
• Kidney disease
• Polyuria: Urine Output > 2.5 L/day
• Increased water ingestion, diuretics
• Uncontrolled Diabetes mellitus and insipidus.
• Anuria: Urine output < 100ml/day
• Kidney failure

• Obstruction such as kidney stone or tumor

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Color:

 Color intensity of urine correlates to concentration, darker color means more

concentrated sample.

 Amber yellow: Due to presence of Urochrome (a pigment that is derived from

urobilin, produced during bilirubin degradation, found in normal urine).

 Colorless: Diluted non-concentrated urine.

 Reddish brown: Blood (Hemoglobin).

 Yellow foam: Bile or medications.

Cloudy: If the sample contains many red blood cells, it would be cloudy as well as red.
Turbidity or cloudiness may be caused by excessive cellular material or protein in the42
urine
Odor:
1.Aromatic odor: Normal urine due to aromatic acids.

2.Ammonia odor: On standing due to decomposition of urea.

3.Fruity odor: Diabetes due to the presence of ketones.

❖ Urine does not smell very strong, but has a slightly "nutty" odor. Some
diseases cause a change in the odor of urine.
❖ For example, an infection with E. coli bacteria can cause a bad odor.

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pH:
 pH measure acidity or alkalinity of urine

 Normal urine pH: 6 - 7.5.

 Increased alkalinity in urine: UTI and kidney stones.

 Sometimes the pH of urine is affected by certain treatments.

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 SPECIFIC GRAVITY
(SP.GR.)
 It is measurement of urine density which reflects the ability of the
kidney to concentrate or dilute the urine relative to the plasma
from which it is filtered
 High
sp.gr. means more solid material is dissolved in the urine
 Measured by: urinometer, refractometer, dipsticks

 Increase in Specific Gravity: Low water intake, Diabetes

mellitus, Albuminuria, Acute nephritis.

 Decrease in Specific Gravity: Absence of ADH. 45

CHEMICAL ANALYSIS OF THE URINE:
The chemical analysis of urine is undertaken to evaluate the levels of the
following components:

• Protein
• Nitrite
• Glucose
• Ketones
• Bilirubin
• Urobilinogen

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CHEMICAL ANALYSIS OF THE URINE:
HOW TO DETECT ABNORMAL CONSTITUENTS:
Urine strip:
Glucose

 Strip is filter paper or plastic which has chemical substance Bilirubin

(reagent) coat on different pads. Ketones

Specific Gravity

 It gives color when react with substance in urine. Blood

pH

 The produced color is compared with a chart color visually or Protein

mechanically assessed. Urobilinogen

Nitrite

Leukocyte

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Results are reported as:
 In concentration (mg/dl)

 As small, moderate, or large

 Using the plus system (1+, 2+, 3+, 4+)

 As positive, negative, or normal

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Urinalysis test strip Automated Urine Testing
Machine
 This method is rapid, easy, give early indication and qualitative.

 Therefore, usually there are other confirmatory tests: (chemistry, microbiology and
microscopic analysis).

 Reaction in strip is affected by time, to reduce timing errors and to limit variations in
color interpretation; automated instrument is used to read the reaction color on each
test pad.

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Strip include the tests:
 Glucose

 Bilirubin

 Ketone

 Specific Gravity

 Blood

 Protein

 Urobilinogen

 Nitrite

 Leukocyte

 pH

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1- Proteinurea:

 It
is the presence of abnormal amount of protein in urine.
 Urine of healthy individual contains no protein due to:

✓ In normal physiology, small M.wt. proteins are reabsorbed by kidney

tubules (proximal tubule)
✓ large M.wt of protein can't pass through kidney tubule to urine. unless
kidney tubule damaged.
 The main protein in urine is albumin therefore,

proteinuria = albuminuria
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Microalbuminuria:
 It is the presence of small amount of albumin in urine.

 Microalbuminuria (defined as urinary albumin excretion of 30-

300 mg/day, or 20-200 µg/min).
 Itis very important in detection of early stage of nephropathy
and in diagnosis of DM complication (nephropathy).
 High protein in urine makes urine looks foamy.

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2- Glucosuria:

 Itis the presence of abnormal conc. of glucose in urine .

 Normally, glucose is reabsorbed by active transport in proximal tubule
and therefore doesn't appear in urine.
 If the blood glucose level exceeds the reabsorption capacity of kidney
tubules (renal threshold), glucose will appear in urine.
 Renal threshold of glucose: is 180 mg/dl.

 Glucosuria indicates that glucose concentration in blood exceeds

this amount and the kidneys are unable to reabsorb it efficiently.
 Causes of glucosuria:

Pregnancy, liver diseases, hormonal disorders, diabetes mellitus. 53


3- ketourea:
 It is the presence of abnormal amount of ketone bodies in urine.

 Body normally uses carbohydrates as source of energy.

 If carbohydrate source depleted or there is a defect in carbohydrate

metabolism, body uses fat as a source of energy.

Fat Fatty Oxidation H2O+CO2+energy

Acids

 When fat oxidation occurs, fatty acids utilization results in production of

intermediate substances (ketone bodies).
 Three ketone bodies: acetone, acetoacetate, b-hydroxybutyric acid

 Elevated levels of ketone bodies in blood and urine cause acidosis which
leads to coma and death.
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Ketonurea is common in uncontrolled DM (why?)

 Because diabetic patient has high blood glucose but can't use by cells,
so lipids are used as source of energy.

 Ketourea present in:

❑ Disease
❑ Nutrition(unbalance diet)
❑ Vomiting for long time

Normal values: No ketone bodies in normal urine.

Small: < 20 mg/dl Moderate: 30-40 mg/dl large > 80 mg/dl 55
4- Hematurea:

 It is the presence of red blood cells (RBCs) in the urine.

 Can’t detected by the naked eye so detection by strip or microscope

 Positive result may be due to stones or tumors.

5- Hemoglobinuria:
 Presence of heamoglobin in urine due to rupturing of RBCs

 This may occur in malaria, hemolytic jaundice.

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6- Bilirubin (Bile):

 Results from hemoglobin breakdown

 It indicates an excess in the plasma.

 Commonest cause of positive results is liver cell injury e.g.

hepatitis, paracetamol overdose, late-stage cirrhosis and
jaundice (biliary duct obstruction).

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7- Nitrite:

 This test is used for screening about presence of bacteria.

 Normal urine contains nitrate but not contain nitrites.

 In the presence of bacteria, the normally present nitrate in the urine is

reduced to nitrite.

 Positive test indicates presence of more than 10 organisms/ml.

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8- Urine leucocytes:
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

WBCs - ≤2-5 WBCs/HPF

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MICROSCOPIC EXAMINATION
1-RBCs
2- WBCs
3- Epithelial cells
4- crystals

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 EPITHELIAL CELLS

 Renal tubular epithelial cells, contain a large round or oval nucleus

 However, with nephrotic syndrome and in conditions leading to
tubular degeneration, the number sloughed is increased.

 ≤15-20 squamous epithelial cells/HPF

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URINE MICROSCOPIC CRYSTALS

Gout

Kidney problem

UTI

Kidney stones

Kidney stones and

dehydration
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Cystinuria