PATHOLOGICAL ANALYSIS
Dr. Wafaa S. Al wazni
PATHOLOGICAL ANALYSIS
-
Histopathology analysis
General analysis
Bacteriology analysis
Serology analysis
Semen analysis
Biochemistrys and Hematology analysis
First \\ Histopathology analysis
 Disease
Is in abnormal condition of a living thing, pathology is that branch of biology
that involves the study of living things in their abnormal forms and conditions is
the development and the structural and functional changes produces by them.
The pathologist acts a consultant to physicians in diagnosis of disease during
life and after death by the laboratory chemistry microbiology hematology
examination of surgical removed specimens and autopsy or postmortem
examination
Individual + disease = Illness
 Etiology
Refer to the cause of disease and contributing factors.
(Ecological, Immunological, Physiological)
 Pathogenesis
Refer to the mechanism of development of disease and it is the ability of M.O to
cause disease by ability to initiation of the infection process and the mechanism
that lead to the development of signs and symptoms of disease.
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PATHOLOGICAL ANALYSIS
Dr. Wafaa S. Al wazni
 Lesion
The characteristic change in an organism produced by disease is a tissue cellular
or molecular alternation that develops as a result of disease-producing or
pathogenic agent ex. Sickle-cell which is due to abnormal hemoglobin molecules
with abnormal pattern of amino acid in the protein lobar pneumonia tumor of the
lung, hemorrhoid, boil, hemolytic disease of the new born (coated RBC).
 Cellular adaptation
The changes that are intermediate between the normal cell and injured cell. The
most important changes of cellular adaptation are:
1. Induction of endoplasmic reticulum development of more endoplasmic
reticulum EPR due to drug administration over a period of time. Ex: increase
amount of (EPR) in liver can detoxify many drugs.
2. Atrophy : is shrinkage of cell by loss of cell substrate or decrease in the size
of organ due to decrease in the size of cell and a trophy is the partial or
complete washing a way of a part of the body.
Causes of a trophy include:




Poor nourishment
Poor circulation
Loss of hormonal support
Loss of nerve supply to the target organ
Atrophy is a general physiological process of reabsorption and breakdown of
tissue involving apoptosis on a cellular level. When it occurs as a result of disease
or loss of trophic support due to other disease or loss of trophic support due to
other disease. It is termed pathological atrophy.
3. Hypertrophy
An increase in size of the cell lead to increase in size of organ new cell are not
formed there is enlargement of cell. Ex: muscles of body bilders.
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PATHOLOGICAL ANALYSIS
Dr. Wafaa S. Al wazni
4. Hyperplasia
An increase in number of cell in an organ or tissue due to mitotic activity that
lead to increase in size or volume. Ex: breast during pregnancy (physiological)
and lactation thyroid hyperplasia (pathological)
5. Hypoplasia,
Failure of an organ
To reach full adult
size (lung kidney)
Aplasia, Agenesis.
total failure of
an organ to develop
6. Metaplasia
Is a reversible change in which one adult type is replaced by another adult cell
type as a result of chronic irritation. Ex: pseudo stratified ciliated columnar
epithelium in tracheobronchial tree to stratified squamous epith. In the habitual
cigarette smoker.
 Cell injury and response
The cell is the basic functional unit of the organism all abnormalities in the
normal processes of the body that result from injury or disease must originate in
the cell.
The causes of injury:
1. Physical: trauma heat cold radiation electrical energy Ex: burning freezing
cell or effect on the molecular structure.
2. Chemical agents: acids, alkalies, poisons may destroy the membranes, alter
cell functions or may cause death.
3. Living agents, bacteria viruses, Rickettsiae and parasites.
4. Nutrition disturbance deprivation in proteins or vitamins.
5. Genetic defects-hereditary disease or mutation during development.
6. Disturbance in arterial circulation-loss of blood supply.
7. Derangement in the immune mechanism-Ag stimuli.
8. Regressive changes in brain muscle and gonads (testes and ovaries)
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PATHOLOGICAL ANALYSIS
Dr. Wafaa S. Al wazni
Reversible cell injury and degeneration:
Degeneration : changes that occur in the cell following injury.
The most common reaction to cell injury are swelling with or without appearance
of abnormal substance in the cytoplasm. They may still be reversible if the
nucleus remain unimpaired and the injurious agent is removed or destroy before
nuclear damge (changes are found in the cytoplasm may be reversible.
Types and reversible degeneration:
a) Cellular swelling : in acute infection or poisoning is the most common type of
acute degeneration,
M\S: the cell appear swollen their outlines may not be clear there is
damage to the plasma membrane and disruption of mitochondria. Best seen in
parenchymal cell of heart kidneys and liver.
b) Hydropic degeneration : great cellular swelling due to highly absorption of
water by the cell.
M\S: fine or coarse vacules in the cytoplasm. Best seen in epithelial cells of
tubules of kidneys.
c) Fatty change : accumulation of fatt in the cytoplasm
M\S: a small membrane - bound inclusion closely to the endoplasmic
reticulum, small vacuoles in the cytoplasm accumulate to form large clear
space. The vacuoles displace the nucleus to the periphery. Often seen in liver,
kidney and heart.
d) Hyaline degeneration : structurless, smooth, homogenous, glassy appearance,
stains pink with hematoxylin.
Ex: old scar tissue - yellow fever in liver, or cirrhosis of the liver.
e) Amyloidosis : proteinaceous material that accumulate extra cellular in tissue
and organs.
f) Mucinous degeneration : carbohydrate and protein, appear blue. Ex: catarrhal
inflammation - epithelial tumor cell.
g) Mucoid degeneration : change that occur in connective tissue defer from
mucinous deg. By higher sulfer content.
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PATHOLOGICAL ANALYSIS
Dr. Wafaa S. Al wazni
 Irreversible cell injury (Necrosis)
It is the cell or tissue death in the living body & resulting from the temporary or
permanent loss of the blood supply to the tissue.
Cytoplasmic degeneration followed by loss of cell structure fragmentation of
organelles & liquefaction of the cytoplasm and nuclear change necrosis is less
orderly than apoptosis, which is part of programmed cell death.
In contrast with apoptosis, clean up of cell debris by phagocytes of the I.S is
generally more difficulty as the disorderly-Death generally dose not send cell
signals which tell nearby phagocytes to engulf the dying cells this lack of
signaling makes it harder for the immune system to locate and recycle dead cells
which have died through necrosis than if the cell had undergone apoptosis. The
release of intracellular content after cellular membrane damage is the cause of
inflammation in necrosis. Causes include: physical, chemical, bacterial or viral,
Prolonged exposure to injury, infection, comcer, poisons & inflamma.
Types
a. Coagulation necrosis loss of cellular details Ex: heart infarction.
b. Liquefaction nec. – softening & Liquefaction of the dead tissue Ex: infarcts of
the brain.
c. Caseous necrosis – soft cheesy appearance. Ex: Tuberculoses necrosis.
d. Gummatous nec. – not soft, but rubbery and firm. Ex: Tertiary lesion of
syphilis.
e. Fat necrosis – associated with pancreatic injuries or disease while spots in the
tissues, foci surrounded by RBCs + PMN later become seat of Ca++
deposition.
f. Gangrene – caused by cuts off the circulation suddenly in diabetics from
atherosclerosis, by trauma, pressure, freezing or infection by Cl. Perfringens.
g. Zonal necrosis : the injurious agent effect a certain zone. Ex: phosphorous &
ca tetrachloride cause zonal necrosis of the lobule of lymph node.
h. Focal necrosis : minute foci of dead tissue – Ex: lesion found in liver in
typhoid fever.
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PATHOLOGICAL ANALYSIS
Dr. Wafaa S. Al wazni
 Inflammation
Is the local reaction of the living body to any injury. It is a well – coordinate
series of vascular and cellular reactions occurs at site of injury
The object of these reaction is:
a.
b.
c.
d.
e.
To destroy or remove the injurious agent.
To limit its spread.
To neutralize toxins.
To remove the remnants of destroyed tissue.
To prepare the area to the final repair of the damage.
Etiology (causes):
Physical → heat, radiate
Chemical → Alcohol, acids
Mechanical → cut
Mo. → Bact
Exudates
Inflammatory edema fluid, highly protein content, high cell counts, low glucose
content, some times clots due to fibrinogen content, its function is to dilute toxins,
brings various types of Abs, and limitation of some infection.
Classification of inflammation:
1. According to duration: Acute, Sub acute, chronic.
2. According to type of exudates:
a. Serous : Watery inflame – edema fluid.
b. Fibrinous containing large amount of fibrinogen which ppt.
c. Catarrhal : Mucus.
d. Purulent or supportive : pus in large amount.
e. Hemorrhagic : RBCs + exudates.
f. Fibrinopurulant – Fibrinogen + PUS
g. Mucopurulent : mucin + PUC.
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PATHOLOGICAL ANALYSIS
Dr. Wafaa S. Al wazni
Inflammatory Cells:
Are derived from leukocytes of blood and certain tissue cells. The normal
proportion of WBCs in blood are:
Neutrophils
Basophiles
54% - 73%
Monocytes
24% - 8%
0% - 1%
Eosinophils 2% - 4%
Lymphocytes 21% - 35%
Bloods cells:
1) Granulocytes : polymorphnucleur leukocytes
a. Neutrophils :- are the first cells to accumulate in acute inflammatory
response, directed by chemotactic agents, they are actively phagocytic,
they are the chief cells in pus.
b. Eosinophils :- phagocytic cells, respond chemotactically to split C3 &
C5 – seen in allergic responses and in healing phase of infl.
c. Basophiles :- they have met achromatic granules that contain heparin
and histamine.
2) Monocytes and Macrophages : both are phagocytic, taking up large & small
particles, dead cells, cellular debris and erythrocytes – kill the organism they
ingest – Monocyte leave the blood stream to become tissue macrophages.
3) Lymphocytes : small round cells, less motile related to immune system.
Tissue cells:
1) Mast cells : similar to basophiles, they contain heparin & histamine.
2) Histiocyte or tissue macrophages : highly phagocytic especially when
particulate matter is to be removed, like carbon particles, part of necrotic cell
and M 0.
3) Giant cells : multinucleated cells may contain 50 or more nuclei found in
tissue formed by fusion of histiocytes.
4) Plasma cells : not normally found in blood, not phagcytic, produce large
quantities of Igs.
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PATHOLOGICAL ANALYSIS
Dr. Wafaa S. Al wazni
Acute inflammation signs:
Swelling, heat, redress, pain and disturbance of function.
Sequence of events in acute inflammation
- Increase in permeability of venules and capillaries.
- Exudation of fluid rich in proteins – albumin and globulins, followed by
fibrinogen – between the junction of endothelial cell of venules.
- Concentration and packing of erythrocytes.
- Slowing of blood stream, stasis.
- Emigration of neutraphils first, monocytes seconds, then others aided by chemo
taxis.
- Accumulation of WBCs and fluid in area of irritant.
- Phagocytosis of irritants by neutrophils & monotypes of M.O
- Reversal of vascular changes.
- Fluid reabsorbed by venules and by lymphatic drainage.
- Repair by ingrowths of capillaries and fibroblasts.
Repair
Is the process by which the tissue returns to normal or approximately normal
state. Cells that have been destroyed are either replaced by healthy cells of the
same type growing in from adjacent living tissue (Regeneration) or by the
replacement of dead cells by fibrous tissue and new blood vessels that also come
from uninjured neighboring tissues (granulation tissue or scar tissue). Is one of
the end stage of inflame.
Microscopic features of a cute inflammation :The acute inflame – lesion shoes varying proportion of :
1. Serous exudates.
2. Fibrin.
3. PMN leukocytes.
4. large mononuclear cells.
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PATHOLOGICAL ANALYSIS
Dr. Wafaa S. Al wazni
According to the character of the exudates, acute in flam : are
classified as follows:
1. Acute serous or serofibrinous inflame : Occurs on serous membranes such as
pericardium, pleura or the peritoneum.
It may be absorbed completely or organized by the ingrowths of connective
tissue.
2. Purulent : predominance of pus cells PMN, it may localized to form an :
a. Abscess, which heals by walling off fibrous tissue and scar formation.
b. Diffused to the surrounding tissue and invade blood stream
(Pyemia) – often caused by streptococci, pneumococci.
3. Hemorrhagic : exudates contains large numbers of erythrocytes due to
capillary injury and rupture, (e.g. hemorrhagic cystitis).
4. Catarrhal :- relatively mild, seromucinous exudates on the mucus membrane,
later becomes mucopurulent.
Ex: in flam of the upper respiratory or alimentary tract.
5. Pseudo membranous or diphtheritic in flam : Is formed by fibrinous
exudates over a necrotic layer of mucosa. Ex: in diphtheria, in alimentary tract
of person receiving large quantities of some antibiotics, and in infection with
resistant staph.
Chronic inflammation
Is a process in which exudative changes are prolonged and proliferation of
connective tissue is the dominate feature of the raction. The cellular of reaction
consist primarly of lymphocyte, plasma cell and macrophages together with
newly formed collagenous fiber. This chrortic process often follows an scute
inflammatory one. When pus associated with chronic lesion, it is termed chronic
supportive inflammation (chronic supportive osteomyelities). The proliferative
activity lead to the formation of abundant scar tissue, which may in itself harm
full. Ex: chromic nephritis where progressive glomerular scaring result due to
functional failure of the kidney. Other cells may involved in chromic inflame :
histiocytes, fibroblast, zosinophils + Basophiles and Giant cells.
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PATHOLOGICAL ANALYSIS
Dr. Wafaa S. Al wazni
Inflammation and cancer
Some types of cancer arise from tissue irritation. And inflammation in fact, in the
body, inflammatory cells such as neutrophils can play the role of turncoat by
helping tumor cells multiply and spread. Some tumors have been likened to
"Wounds that wont heal".
Melanoma, which is caused by too much exposure to ultraviolet light from the
sun, is thought to be caused in part by uncontrolled inflammation of the skin.
In recent years, scientists have unveiled another link between inflammation and
cancer.
Medicine that block inflammation, such as aspirin, Tylenol and other so – called
non-steroidal anti-inflammatory drugs (NSAIDs), have been shown to prevent
certain kinds of colon, lung, mouth and stomach cancers.
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PATHOLOGICAL ANALYSIS
Dr. Wafaa S. Al wazni
Second \\ General analysis includes:
 General Urine Examination (G.U.E):
Urine is a complex aqueous mixture consisting of %96 water +4% dissolved
substances, most of which either are derived from the food eaten or are waste
products of metabolism.
The dissolved substances consist primarily of urea (the principle end products of
protein metabolism, Nacl, sulfate and phosphates).
This dissolved substances in Normal urine may be divided into:
1. The normal organic substances includes: Urea, Uric acid and Creatinine.
2. The normal inorganic substances include:
(Cation: Na+, K+, NH 4 )
(Anion: Cl  , PO 43 , Sulfate SO 4 2 )
3. Normal urine Specimen also contains certain formed elements in low
concentration, like Red blood cells, Pus cells and epithelial cells.
 Cellular Constituents
I) Red Blood Cell
RBCs are abnormal urinary constituents and the presence of more than two per
high power field (H.P.F) is always of pathological significance. The condition in
which RBCs are found in the urine is termed as hematuria. The distinguish
between hematuria and hemoglobin uria that the last one mean presence of free
hemoglobin in the urine. The degree of hematuria may vary from a frankly bloody
specimen on gross examination to a specimen that show no change in color.
II) Leukocytes (white blood cells, pus cells)
The presence of a few leukocytes in urine is normal. More than an occasional cell
(1-5/H.P.H) is considered abnormal.The presence of large number leukocytes in
the sediment indicates inflammation at some point a long the urogenital tract.
The inflammation may result from a bacterial infection or other causes.
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PATHOLOGICAL ANALYSIS
Dr. Wafaa S. Al wazni
III) Epithelial Cells (E.C)
The structures that make up the urinary system consist of several layers of E-C,
except for the single-layered tubules of the nephron.
The E.C of organs such as the urethra and bladder (besides the contaminating
cells of the male and female genital tracts) are continually sloughed off into the
urine and replaced by cells originating from deeper layers. Therefore, urine
always contains some E-C.
IV) Casts
Casts are long cylindrical structures that result from the solidification of material
within the lumen of the kidney tabules. They are important because anything that
is contained within the tubules is flashed out in the casts. Casts may be formed at
any point of the nephron, either by precipitation of protein or by grouping
together of material within the tubular lumen.
Since cast represent a biopsy of the kidney, they are extremely important
clinically. They are often contain RBCs., E-C, fat globules, bacteria and
leukocytes.
The types of the casts:
1) Hyaline cast
2) Granular casts
3) Blood or RBC casts
4) Leukocytes casts
5) Tubular epithelial casts
6) Waxy casts
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PATHOLOGICAL ANALYSIS
Dr. Wafaa S. Al wazni
When give normal result to (G.U.E):
1. The color of urine varies from pale to deep amber.
2. The specific gravity range between 1.008-1.030.
3. The appearance of the urine is clear. On standing Ammonia maybe formed by
bacterial decomposition of urea, the resulting decreases of acidity which may
cause cloudiness due to precipitation of calcium phosphate.
4. Contains minute traces of protein and sugar that not detect by routine methods.
5. Contains a few sequamous epithelial cells.
6. Urea found in rate about 2 mg/l
When give abnormal results to (G.U.E):
1. Contain red blood cells (hematuria).
2. Contain white blood cells. The presence of large numbers of leukocytes in the
sediment indicates inflammation which may results from bacterial infection or
other causes.
3. Contain epithelial cells which results from continually sloughed of E.C into the
urine and replaced by cells originating from deeper layers.
4. Contain Casts.
How can make G.U.E?
1. Take urine specimen
Ideal specimen for microscopic analysis of the urinary sediment is a fresh &
first morning specimen, because it is most concentrated and is particularly
important for reliable results.
Several changes that may occur as the urine stands which are include:
 Red blood cells become distorted because the lack of an isotonic solution.
 White blood cells disintegrate in hypotonic solution
 Cast disintegrates, especially as the urine becomes alkaline because they
must have sufficient acidity & solute concentration to exist.
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PATHOLOGICAL ANALYSIS
Dr. Wafaa S. Al wazni
2. Gross examination of urine
Physical properties of urine
Urinalysis usually involves an assessment of physical properties, such as volume,
colour, transparency and specific gravity.
1st) Volume
The volume of the urine is not measured as a part of a routin urinalysis. However,
in certain conditions the volume of urine excreted in 24 hours is a valuable aid to
clinical diagnosis. In normal adults with normal fluid intake, the average 24 hours
urine volume is 600-1600 ml. under normal conditions, there is a direct relation
between urine volume and water intake.
Polyuria : The consistent elimination of an abnormally large volume of urine,
over 2 L in 24 hours.
Oigouria : The excretion of an abnormally small amount of urine less than 500
ml in 24 hours.
2nd) Colour
The normal colour of the urine varies from pale yellow to deep yellow, the
normal colour of urine seem to result from the presence of three pigments:
urochrome, uroerythrin and uribilin. Urochrome is a yellow pigment and is
present in larger concentrations than other two.
Uroerythrin is a red pigment and urobilin is an orange-yellow pigment.
Colour of urine and disorder:
1) Pale urine may be associated diabetes mellitus or diabetes insipid us. The first
one with sugar and high specific gravity. Also pale urine seen with a large
quantity of protein in the nephritic syndrome.
2) Dark yellow or brown-red indicate of very concentrated constituents and low
volume. It is ofyen seen in fever.
3) yellow-brown indicates the presence of bilirubin.
4) Drange-red or orange-brown indicate urobilin or urobilinogen .
5) Clear red is characteristically contains hemoglobin .
6) Cloudy red is characteristically the presence of RBC.
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PATHOLOGICAL ANALYSIS
Dr. Wafaa S. Al wazni
3rd) Appearance
When voided, urine is normally clear and most urines however, will become
cloudy when allowed to stand.
The causes of cloudiness may be solid materials that will be visible under the
microscope. There are numerous words have been used to descried the degree of
transparency of a urine specimen like, clear, cloudy, very cloudy and turbid.
The causes of the cloudiness are:
1) On standing cloudiness may result from the presence of mucin, or mucous
threads in the urine.
2) Inflammatory state of the lower urinary or genital tract.
3) Amorphous phosphates and occasionally carbonates.
4) Bacteria are another common cause of cloudiness in urine specimens that have
been allowed to stand. In this case bacteria are not clinically significant.
5) Spermatozoa or prostatic fluid may also cause cloudy.
6) Amorphous urate in acidic urine.
7) WBC or pus will be seen as white cloudiness.
8) RBC and vaginal contamination.
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PATHOLOGICAL ANALYSIS
Dr. Wafaa S. Al wazni
4th) Specific gravity
The kidney is a regulator of the volume, acidity, composition and osmotic
pressure of the extracellular fluid. A measurement of the specific gravity of urine
is one means of assessing the ability of the kidney to regulate the composition and
osmotic pressure of the extracellular fluid.
Specific gravity of urine is a measure of the amount of dissolved substances
present. More technically specific gravity describe the weight of a solution
compared to the weight of an equal volume of water. It is the ratio of the density
of the solution to the density of pure water density is the mass or weight of a
substance per unite volume.
Clinically, the S.G of urine may be used to obtain information about two general
function the state of the renal epithelium, and the state of hydration of the patient.
The normal value of S.G is 1.008-1,030. However, if the renal epithelium is not
functioning adequately, it will gradually lose the ability to concentrate and dilute
urine.
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PATHOLOGICAL ANALYSIS
Dr. Wafaa S. Al wazni
 General Stool Examination (G.S.E) :
Brown soft mass is normal stool specimen. The normal color influenced by diet &
the formation of this color requires bacterial oxidation to take place in the Colon.
The brown color of stool is caused by stercobillin (pigment derived from bilirobin
after conversion to arobilin).
When give normal result to (G.S.E)
A soft but formed stool specimen is normally seen with brown color.
When give abnormal or pathological results to (G.S.E)
1. Seen yellow to yellow green color: This indicates found diarrhea or the normal
bacterial flora are not present in the bowel as in antibiotic therapy.
2. Appear clay color: occur when stercobilin is absent & there is some increase in
fat.
3. The specimens are bulky, torthy, foul- smelling and found abnormally in large
amounts of fat in the faeces, this case associated with cystic fibrosis and is
called steatorrhea.
4. Dark specimens may be seen after the ingestion of iron and formed when
bleeding from the upper gastrointestinal tract& the bloody specimen usually
associated with disease of the lower rectum & anus.
5. Mucous & pus seen in faeces associated with inflammatory conditions such as
colitis or a bowel tumor but pus only seen in disease such as ulcerative colitis
& bacillary dysentery.
6. In faeces may be seen adult round worms or segments of tape worms
macroscopically. The most important intestinal parasitic infection of
human are:
 Class: Amoebae, Entamoeba histolytica
 Class: Flagellates, Giardia lamblia
 Class: Ciliates,
Balantidium coli
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PATHOLOGICAL ANALYSIS
 Class: Sporozoa,
Dr. Wafaa S. Al wazni
Isospora haminis
Third \\ Bacteriology analysis :
A. Infection of wounds &other tissues
B. Infection of Blood
C. Infection of CSF
D. Infection of upper respiratory tract infection
E. Infection of lower respiratory tract infection
F. Infection of Gastrointestinal tracts
G. Infection of Urinary tract
A. Infection of wounds & other tissues
There are many types of these infections are:
 Nosocomial wound infection
Ex: Staph aureus
Pseudo aeranginosa
Coliform bacilli
 Soft tissue infection:
Ex: many of aerobic & anaerobic bacteria
 Burns:
Ex: S. aureus , P. aeroginosa
 Bone infections ( osteomyelitis)
- S. aureus & coliform bacilli in infants
- Group A Streptococci & H. influenzae in children
- Various G -ve cocci & bacilli in adults.
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PATHOLOGICAL ANALYSIS
Dr. Wafaa S. Al wazni
In order to make the laboratory test for these infections should be
follow these steps:
 Specimens
Pus, exudates or fragments of tissue& pieces of abscess wall, if the patient is
febrile or in shock a sample of blood should be taken for culture.
 Lab Examination include
1. Naked eye examination: The appearance of pus or exudates depended on
colour, consistency& odor.
 The
pus is creamy &
Ex: Staphylococcus lesions.
thick
in
consistency
with
pus
cells.
 The pus is watery & straw colored. Ex: Str. pyogenes.
 The pus with fishy smell. Ex: Proteus infection.
 The pus with a sweat – musty odor & often blue pigmentation.
Ex: Pseudomonas infection.
 The pus has an offensive putrid smell. Ex: Anaerobic organisms’ infections.
2. Microscopy examination:
 Gram stain show bacteria which belong to one of these types:
- G+ve Cocci in clusters (Staph) or in chains (Strep) or Diplococci
(Pneumococci)
- G +ve filaments (Actinomyces)
- G +ve rods large straight (Cl. perifringes: gas gangrene), (B. anthracis:
anthrax)
- G -ve rods (E. Coli, Klebsiella, Proteus, Serratia, Pseudomonas)
 Ziehl - Neelson stain
Mycobacterium Nocardia
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PATHOLOGICAL ANALYSIS
Dr. Wafaa S. Al wazni
 Immuno fluorescent staining with specific antisera for some pathogenic
Clostridia.
3. Culture
After taking specimen from a patient, should be cultured on one type of these
media:
CULTURE
MEDIA
Blood agar
Cook meat
broth
Thioglycocte
broth
Macconkey
agar
Sabouraud
dextrose
agar
Lowenstein
jensen
media
Aerobically
(in 5-10%
CO2)
Blood agar
G –ve rods
Yeast
Acid fast
rods
-Coaqulase
-Mannitol
salt agar
Anaerobically
In N or H
5-10%
CO2
Biochemical
tests
Staph
Clostridium
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Mycobacterium
PATHOLOGICAL ANALYSIS
Dr. Wafaa S. Al wazni
B. Infection of Blood (Bacteraemia)
The blood is sterile fluid, does not contain any M.O or toxic substances.
The transient reach of fungi or bacteria to the blood and the blood serves as site of
presence of bacteria, but the bacteria usually cleared from the vascular system
with no harmful effect this called bacteriamia or fungimia, but the condition in
which blood serves as a site of bacterial multiplication as well as a mean of
transfers of infections a gents fromone site to another, this called septisemia.
The most common causes of these infections are:





G +ve Cocci .Ex: S. aureus, S. epidermiles
G -ve Cocci. Ex: N. meningitidis
G +ve bacilli. Ex: L. monocytogenes Clostridium
G -ve bacilli. Ex: Enterobacterasea family
Fungi. Ex: Candida albicans, Cryptococcus neoformans
Specimens :
10 ml of blood for adults, 2-5ml for children and 1-2 ml for infants is transported
to the lab in a tube containing anticoagulant solution or added to the blood culture
bottles.
Note: One to 3 blood cultures should be taken separated by 1hour intervals or less
if treatment cannot be delayed, so that the chance of missing a transient
bacteraemia is reduced and the pathogenic role of S. epidermidis is confirmed.
Lab Examination (Blood culture)
1. The blood should be mixed with 10 times its volume of broth (5 ml blood in
50 ml broth which is tryptic soy broth (TSB) to dilute any antibiotic present
and to reduce the bactericidal effect of serum.
2. Incubated the bottle at 37°C for 7 days. A sterile culture shows a layer
of sedimented R.B.Cs covered by a pale yellow transparent broth, but
microbial growth is evidenced by a floccular deposit on top of the blood
layer. Turbidity, haemolysis, coagulation, gas production and white grains on
the surface or deep in the blood layer.
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PATHOLOGICAL ANALYSIS
Dr. Wafaa S. Al wazni
Blood culture bottles after
Incubation for 24h at 37°C
Gram stain and
Subculture
G –ve rods
MacConkey agar
Biochemical test for
detection of bacterial
genesis
G+ve Cocci
Staph
Strept
Mannitol salt agar
Blood agar with
optochin, tellurite&
bacitracin discs
CAMP test (Bile
esculine agar)
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PATHOLOGICAL ANALYSIS
Dr. Wafaa S. Al wazni
C. Infection of CSF (Meningitis)
CSF is sterile, clear and color less, contains 0-5 leukocyte/mm3 & no RBC.
There are 2 types of meningitis which are:
1. Purulent meningitis (CSF is turbid & contain 100-3000 PMNs/ mm3 )
Ex: H. influenzae
N. meningitidis
In all age groups
But in infants (to 2 months) Ex: E. coli
Salmonella spp
L. monocytogenes
2. A septic meningitis (CSF is clear or slightly turbid, contains 10-500
leucocytes/mm3, mostly lymphocytes). Ex: Candida albicans
Leptospira
Specimens
1. 3-5ml of CSF is collected in two sterile tubes, one for chemical examination
(glucose& protein), the other for microbiological examination & leukocytes
count.
2. Blood culture should be collected because meningitis is often associated with
bacteraemia.
3. The appearance of CSF should be noted: Clear, turbid, purulent, yellow
(previous hemorrhage), contamination with blood.
Lab Examination
 Microscopic examination which represented by:
a) Direct examination for leukocytes, RBCs, Bacteria, yeast.
b) Gram stain: very important because the culture depend on its result.
c) Acid fast stain for tuberculosis meningitis.
 CSF culture
As shown in this diagram:
23
PATHOLOGICAL ANALYSIS
Dr. Wafaa S. Al wazni
24
PATHOLOGICAL ANALYSIS
Dr. Wafaa S. Al wazni
CSF
G+ve Cocci
CAMP test
& optochin
sensitivity
for Strep.
Blood agar
Chocolate
agar
G –ve rod
G –ve rod
N.
meningitidis
Catalase test +ve,
motility +ve, bile
esculin agar (growth&
black discoloration)
for Listeria
monocytogenes
Macconkey
agar&
biochemical tests
for
enterobaceriaceas
Oxidase
test
Slide aggl
25
Blood agar
with
S. aureus
Lowen stain
Jensen media
H. Influenzae
(Satellite
colonies)
Tuberculosis
Slide aggl
PATHOLOGICAL ANALYSIS
Dr. Wafaa S. Al wazni
D. Upper respiratory tract infection :
The Upper respiratory tract extends from the larynx to the nostrils and
communicating cavities the sinuses & the middle ear.
There are many types of infection:






Pharyngitis(sore throat)
Nasopharyngitis
Otitis media
Sinusitis
Epiglottitis
Diphtheria (laryngitis & tracheitis)
Diphtheria
One of the important diseases caused by C. diphtheria which spread by droplets
or by contact. The bacteria multiply locally without invading deeper tissue or
spreads through the body. The toxin destroys epithelial cells & PMN & an ulcer
from which is covered with necrotic exudates forming a false membrane (pseudo
membrane) over the tonsil pharynx & within the membrane produce toxin which
is absorbed into the lymphatic & blood, it cause fever pallor, exhaustion,
mycocarditis & polyneuritis with paralysis of eye muscle.
Specimen
Should be collected after the tongue is kept down with a tongue depressor & a
sterile cotton-wool swab is rubbed vigorously over each tonsil over the back wall
of the pharynx & over any other inflammation area.
Direct Microscopy
Gram stained smear& the direct smear has specificity for the detection of the
diphtheria bacillus which appear rod club shape with irregular swell at one end;
but when stained with methylene blue, cell in stained smear tend to be parallel or
at acute angles to one another.
26
PATHOLOGICAL ANALYSIS
Dr. Wafaa S. Al wazni
Culture
The swab should be rubbed over blood agar plate and Loffler coagulate smear
(selective media for C. diphtheria), then incubation for 24h at 37°C. After growth
appears complete other test to detect the type or genus of bacteria.
Elek test
Detection of toxin (Gel diffusion precipitin reaction) by producing serum in
horses.
27
PATHOLOGICAL ANALYSIS
Dr. Wafaa S. Al wazni
E. Lower respiratory tract infection :
All infection occurring below the level of the larynx which involved trachea &
lung tissue; such as:






Epiglottilis
Laryngotracheobronchiotitis
Whooping cough
Acute bronchitis
Acute Pneumonias ( Labor pneumonia )
Chronic chest disease ( Tuberculosis )
Whooping cough
The bronchial & pulmonary secretions of exudates are often studied by
examination sputum; the most misleading aspect of sputum examination is the
almost inevitable contamination with saliva & mouth flora. Thus finding Candida,
S-aureus, even Streptococcus pneumonia in the sputum of patient with
pneumonitis has no etiologic significance unless supported by the clinical picture.
In whooping cough the personal swab in a special transport media is required,
because this disease is highly contagious infection occur by respiratory droplet
contain Bordetella pertussis which causative agent for this infection & not
invade tissue or enter the blood but produce an endotoxin able to destroy the cilia
cells, which in turn allows mucus to accumulate or the M.O attach to & multiply
in the ciliated respiratory mucosa but don’t invade deeper & the surface
component play important role in specific attachment to respiratory epithelium.
28
PATHOLOGICAL ANALYSIS
Dr. Wafaa S. Al wazni
Tuberculosis
Is a chronic disease caused by mycobacterium tuberculosis it most common in
being but may occur in other organ, mode of infection by inhalation of droplets,
(coughing, sneezing).
M/S: appearance of large mononuclear cells, histiocytes, which become the
characteristic large epithlioid cell with vesicular nuclei and abundant cytoplasm.
Organization may occur & result in fibrous scarring in some cases, or aggressive
disease may produce abscess.
Resolution of the exudates usually restores normal lung stricture but this impaired
sometime by organization, scarring & abcess formation.
Lobar pneumonia
This pattern of acute bact. Inflation involves a large portion of or an entire lobe of
the lung.
Most lobar pneumonias are caused by pneumococci, which enter the lung via
airways. Occasionally they are caused by other organisms (Klebsiella,
pneumonia, stash., strep, H. influenza).
The following sequence of stage is ((classic)) but infrequently seen because of
antibiotic therapy. However, the various stages portray the natural history of
uncomplicated lobar pneumonia.
 Congestion
 Predominates in the 1st 24 hours.
 Red hepatization (consolidation): describes lung tissue with confluent acute
exudation containing neutrophils & red cells, giving a red, firm, liver – like
gross appearance.
 Gray hepatization: as red cells disintegrate & the remaining fibrinosuppurative
exudates persist, giving a gray brown gross appearance.
 Resolution is the favorable final stage in which consolidated exudates
undergoes enzymatic & cellular degradation & clearance. After these stages,
normal structure is restored.
29
PATHOLOGICAL ANALYSIS
Dr. Wafaa S. Al wazni
 Collection of sputum specimen:
The sputum should be collected in a sterile container with a secro, & send to the
lab without delay. If the sputum is allowed to stand after collection over growth
of contamination of bacteria may take place before the examination is carried out.
 Lab examination:
Include: Microscopic Examination :
A portion of purulent or mucopurulent sputum should be used for the preparation
of Gram stain smear which give first impression for some M.O responsible for
infection such as S. aureus , H. influenza ( coccoid bacilli, some times occurring
in pairs or short chains ), & Bordetella pertussis in swab (cocco bacilli with
Toludine blue stain, bipolar metachromatic granules can be demonstrated ).
 Culture:
Select the purulent material in sputum or swab & incubate on the various culture
plates which include:
- Blood agar
- Chocolate agar
- Macconkey agar
These agars incubated for 24h at 35-37°C & reincubation for an extra 24 hours
when growth is less than expected; but swab taken from Nasopharyngeal
of patient with whooping cough can culture on Bordet - Genqou medium
(potato-blood-glycerol media) in a moist environment (in sealed plastic bag) or
cough droplets expelled on to a cough plates, held in front of mouth during a
paroxysm.
30
PATHOLOGICAL ANALYSIS
Dr. Wafaa S. Al wazni
F. Gastrointestinal tract infection
Acute diarrhea with or without vomiting is a common complaint. Microbial
causes, either by multiplication in the intestine or from the effects of preformed
toxin, are the most important reasons for acute gastro-intestinal upset in or
otherwise healthy individual.
The common microbial causes of infections intestine disease are :
 Viruses: Rotavirus, Astrovirus, Calicivirus.
 Bacteria: Salmonella Spp., Campylobacter Spp., Shigella Spp., E. coli, Vibrio
cholera, V. parahaemolyticus, Yersinia enterocolotica.
 Protozoa: Cryptosporidium parvum, Entamoeba histolytica, Giardia lamblia.
The GID can be divided into many types:
a) GID associated with toxin producing M.O:
 Enterotoxin: caused by Clostridium botulinum, botulinal toxin after
absorption into the blood stream, the toxin binds to the presynaptic nerve
ending of the peripheral nervous system & cranial nerves, where it inhibits
acetylcholine release.
 Cytotoxin: disrupt the structure of intestinal epithelium, Ex: Shigella
dysenteriae. But in V. cholera produce entrotoxin diarrheal disease. The
bacteria remain within the intestinal tract attach to microvillis of the brush
border of epithelial cells. There the bacteria multiply & liberate Cholera
toxin, that cause rice water stool contain mucus epithelial cell& large
numbers of vibrios without PMN or blood.
b) GID associated with invasive mucous:
Certain parasites, Ex: Entamoeba histolytica invade the colonic mucosa,
producing characteristic ulcerative lesions& a profuse bloody diarrhea
(dysentery). The stool specimen examined by direct microscopy & the
E. histolytica maybe recognized by its active movement, pushing out fingerlike pseudopodia & sometimes progressing across the microscopic field.
31
PATHOLOGICAL ANALYSIS
Dr. Wafaa S. Al wazni
c) GID associated with adherent microorganisms:
Ex: Giardia lamblia, which attached to the mucosal surface of the upper small
intestine & cause malabsorption of fat & chronic diarrhea. Under microscope
the G. lamblia is kite-shaped with two nucleated sucking pads& four pairs of
flagella.
Specimens
Faecal specimens should be collected in the early stages of disease, in a sterile
container & these specimens should be processed as soon as possible & not stand
longer than 2 hours after collection, if it is not possible the specimen collected on
swab & then incubated in a suitable transport medium & then make smear &
stain.
Laboratory examination
 Naked
eyes examination:
The stool specimen should be directly examined to determine:
- The consistency (solid or liquid (diarrhea))
- Color (white, yellow, brown, bloody)
- Typical components (mucus, blood (dark to black))
 Microscopically
examination:
Saline & iodine preparation should be made & examined with low & high power;
various ova may be seen in feces. Ex: - Hook warm
- Ascaris (Round warm)
- Taenia
- Trichuris trichiura
32
PATHOLOGICAL ANALYSIS
Dr. Wafaa S. Al wazni
Culture includes these steps:
 Preparation
of fecal suspension & inoculation on agar plates:
The stool should be suspended in saline to obtain a turbid suspension but liquid
stool do not require the addition of saline.
 Inoculation
of specimen
There are differences for adult & child. For adults: the specimen should be
inoculated on tetra thionat broth for the harvestation of Salmonella & Shigella
from stool because these bacteria found in small number in stool. Then cultured
on MacConkey if appear pink colonies the result is Negative & these colonies
neglected but when other colonies should be transfer to the S.S Agar for detection
of Salmonella & Shigella bacteria, appearance of black colonies indicates
Salmonella but white colonies indicate Shigella.
After that serological identification should be performed. Transfer stool specimen
to peptone water (PH 8.6) after 6-8 hr incubation on TCBS (Thiosulfate- Citratebile-Sucrose) after incubation at 37 °C for 24 hr. Yellow colonies that appear
identified by set of sugar & agglutination test (Vibrio).
In children: growth of any bacteria on the appropriate media is considered as
pathogenic in children. A sample is inoculated on tetrathionate broth, MacConkey
agar, Mannitol salt agar, EMB as shown in this diagram:
33
PATHOLOGICAL ANALYSIS
Dr. Wafaa S. Al wazni
Specimen from
child incubated
to
Tetra thionate
broth
Macconkey agar
Mannitol salt
agar
EMB agar
S.S agar
Pink colonies
Yellow colonies
S. aureus
Green metallic
sheen (E. coli)
Coagulase
Sensitivity to
perform the
identification
Sensitivity test
+ biochemical
test
E. coli
Salmonella &
Shigella
Serology &
sensitivity test
34
PATHOLOGICAL ANALYSIS
Dr. Wafaa S. Al wazni
G. Urinary tract infections :
The urinary tract consists of the kidney, bladder& the urethra. The bacterial
infection is usually acquired by the ascending route from the Urethra to the
bladder& may proceed to the kidney.
The diagnosis of urinary tract infection cannot be made without bacteriological
examination of the urine because many patients with the frequency dysuria
syndrome have sterile urine & conversely asymptomatic bacteriuria is common
condition.
A scending infections of the urinary tract are most commonly caused by G –ve
rod such as E. coli & other members of the Enterobacteriaceae. Klebsiella,
Enterobacter, Serration & P. aeruginosa are more frequently found in hospital
acquired UTI, because their resistance to antibiotics & is the most frequent source
of bacteriamia.
All areas of the UT above the Urethra in a healthy human are sterile, but the
epithelium of the Urethra usually colonized by some normal flora M.O such as
coagulase, negative staphylococcus, not hemolytic streptococcus, lactobacillus
and Diphtheroid; In addition some non pathogenic bacteria such as Neisseria,
anaerobic bacteria& G –ve bacilli.
Most urinary tract pathogen originate in the faecal flora, which have ability to
colonize the periurethral areas because have particular type of pili, enable them to
adhere to Urethral & bladder epithelium and some of them have capsular which
associated with ability to cause pylonephritis.
 UTI is primarily of three types:
 Cystitis
infection of the bladder
 Pyelorephritis
 Uritis
infection of renal parenchyma
infection of women more than men.
35
PATHOLOGICAL ANALYSIS
Dr. Wafaa S. Al wazni
Laboratory work
 Specimens
Mid stream urine specimen collection must be performed carefully for optimal
results especially in females. In some time use urinary catheterization which
allow collection of bladder urine with less Urethral contamination. The
contamination of urine sample by normal perineal & anterior urethral flora is the
most important consideration for collection of clinical samples, contamination
with vaginal discharge may account with vaginal discharge may account for
presence of albumin & pus. Container used for collection of urine samples, should
be sterile & clean.
 Microscopic examination
1. A drop of fresh uncentrifuged urine placed on a slide, covered with a cover
glass, and examined with light microscope can reveal leukocytes, epithelial
cells & bacteria if can more than 105 ml are present.
2. Brief centrifugation of urine readily sediments pus cells, which many carry
along bacteria & thus many help in microscopic diagnosis of infection.
 Culture
The collected urine is cultured in measured amounts on solid media, such as
blood agar plates& other solid media. Then identification of the M.O grows on
these media.
36
PATHOLOGICAL ANALYSIS
Dr. Wafaa S. Al wazni
H. Genital & Sexually transmitted pathogens (STDs)
The number of M.O known to be STD & the laboratory approach to the diagnosis
of genital infection is related to the sex of the patient, although some infections
are common to both sexes. Ex: Gonorrhoea, Syphilis & Chlamydial infection but
there are usually differences in the presenting symptoms & the sites & methods of
specimens collection in these infections.
 General infection in women:





Acute Vaginitis: Candida albicans
Cervisitis with or without Urethritis: Clamydia trachomatis
Uterine Sepsis: S. aureus
Genital ulceration: T. palladium
Tuberculosis of uterus: M. tuberculosis
Laboratory works
Specimens: Vaginal & urethral discharge is collected by swabs made of cotton
that has been treated with charcoal to adsorb toxic material to gonococci,
Mycoplasma & Chlamydia.
 For diagnosis of vaginitis or uterine sepsis the specimen is high vaginal swab
(upper part of vagina).
 For gonorrhoea the vaginal discharge swab is unsuitable because gonococci
tend to die in the acid vaginal secretion and if remaining viable are likely to be
over grown by vaginal commensal bacteria so that endocervical swab must be
collected. Urethral & pharyngeal swabs should be taken.
Swabs for culture should be placed in tubes of Amines transport medium or
modified Stuart’s media & transport to the lab. (Held at room temperature until
inoculation).
 For examination of Trichomonas special specimens should be collected from
the vagina, urethra, cervix, including a swab placed in clear Trichomonas
transport medium or saline for microscopy &culture.
37
PATHOLOGICAL ANALYSIS
Dr. Wafaa S. Al wazni
 Since Chlamydia is intercellular pathogens, it is important to remove epithelial
cells with swabs from the urethral mucosa.
Microscopical examination:
Both a wet film & gram stained film should be examined; the wet film is
examined for the presence of motile Trichomonas vaginalis (rounded or pearshaped):
1. Examine under dark field microscope for T. pallidum
2. The gram stained film should be examined for Candidosis & bacterial
vaginitis.
3. Candida = G +ve yeast forms & G +ve hyphae (Pseudomycelium)
4. Bacterial vaginitis = G –ve bacilli of diphtheroid morphology
The diagnosis of bacterial vaginitis does not depend on the isolation of the
organisms (G. vaginalis or M. hominis) but by:
1. The presence of homogenous vaginal discharge which adheres to the vaginal
wall in a thin film that can vary from white to grey in color.
2. A fishy smell that can be detected by the addition of %10 KOH (NaOH) to
fresh vaginal discharge (amine like odor).
3. Absence of G +ve bacilli (lactobacilli) & replacement with a mixed flora
consisting G. vaginalis anaerobes such as Bacteroides.
4. The presence of clue cells, squamous epithelial cells covered with bacteria.
5. An increased vaginal PH ≥ 5
Culture:
The specimen should be inoculated on two plates of a rich blood agar, one
incubated 35-37°C in %5 CO2 with moisture & the other in anaerobic atmosphere
with CO2.
1. Candida albicans can be recognized on the aerobic blood agar & grow well on
Colombia agar base + %5 sheep blood + Naldixic acid (CAN) & Sabourauds
agar.
2. N. gonorrhoeae grow well on rich blood agar & Thayer – Martin medium
which contains the antibiotics.
38
PATHOLOGICAL ANALYSIS
Dr. Wafaa S. Al wazni
3. For Trichomonas vaginalis the Cysteine Peptone – Liver infusion Maltose
(CPLM) medium is used under anaerobic condition.
 General infection in men:
The infection in men are mostly caused by same organisms as in women,
include:
1. Urethritis:
Is classified as gonococcal or non gonococcal (NGU) depending on whether or
not gonococci are found in gram film culture of discharge.
2. Prostatitis:
Is usually caused by gonococci or Chlamydia.
3. Ulceration:
Caused by T. pallidum.
Collection of specimens & laboratory examination:
Urethral discharge may be expressed directly on to the slide for gram stain & be
inoculated immediately onto chocolate agar & selective medium for the culture of
gonococci.
If specimens have to be transported to the laboratory the exudates from ulcers
should be collected on a swab & put into a tube of Amine’s transport medium.
39
PATHOLOGICAL ANALYSIS
Dr. Wafaa S. Al wazni
Fourth\\ Serology analysis :
Serology: is a science that attempts to detect signs of infection in patient’s serum
such as antibodies (Abs) specific for a microbe.
The basis of serological tests is that Abs specifically binds to Ag in vitro.
The unknown Abs can detect when known Ag is added to serum & the reverse is
true.
The serological tests used the principle of :
A. Precipitation:
It involves the interaction of solute Ag (precipitinogen) with Ab (precipitin)
in correct proportions resulting invisible precipitate. The Ags are solutions of
molecules which are protein or carbohydrate in nature.
T he reaction is carried out in semisolid media like agar gels through which
soluble molecules can diffuse & give precipitation line, this reaction is called
Immunodiffusion test.
B. Agglutination:
It involves the interaction of suspended particulate Ag (agglutinogen) with
their specific Ab so agglutination or clumping of the particles is seen.
 There are many types of agglutination:
1. Active Agglutination or bacterial agglutination
Is used to diagnosis some bacterial diseases like Widal test & Weil Felix test.
2. Hemagglutination
The Ags are parts of cell membrane surface of RBCs & when mixed with their
Abs caused clumping of the RBCs, like ABO system of blood types.
3. Hemagglutination inhibition
The virus reacts naturally with the RBCs & agglutinates them but when the
Abs for the virus are present they attach to the virus & the RBCs remain free
40
PATHOLOGICAL ANALYSIS
Dr. Wafaa S. Al wazni
so there is no agglutination (+ve result). This test is used for diagnosis of
several viral diseases (measles, rubella …etc).
4. Passive agglutination
The Ags adsorbed on the surface of a particle like RBCs latex & bentonite. In
latex agglutination test the polystyrene latex particles coated with the Ags
which are agglutinated with specific Abs like kits used for identifying yeasts
(Candida) & bacteria (Staph, Strept & gonococci).
5. Passive Hemagglutination
The Ags adherence readily on RBCs, (like polysaccharide) or require tannic
acid treatment before adsorption of Ag (protein) on RBCs which used as
carrier.
C. Immunoelectrophoresis:
The Ag are separated by electrophoresis on a gel & to identify the Ags bands,
specific Abs placed in a trough & allowed to diffused toward the bands Ags
& arcs is formed, this test is useful for the differentiation of Ags with in a
mixture & used widely to detect disorders in the production of Abs(serum
protein, albumin, globulin).
41
PATHOLOGICAL ANALYSIS
Dr. Wafaa S. Al wazni
D. The complement fixation test:
The complement fixation test detects Ab.
1. A test antiserum is titred in doubling dilutions & a fixed amount of Ag is added
to each tube or will; if Ab is present in the test serum, immune complexes will
form.
2. Complement is then added to the mixture. If complexes are present, they will
fix complement& consumed it.
3. Indicator cells (red cells) together with a sub agglutination amount of antibody
(erythrocytes cells) are added to the mixture.
If there is any complement remaining, RBC cells will be lysed. If it was
consumed by immune complexes in stage, there will be insufficient to lyse the
red cells.
Antigen
Complement
Test intisera
No imm. Comp.
Free Comp.
Immune Comp.
Indicator
No cell lysis
Indicator cell lysis
42
Fixed Complement.
PATHOLOGICAL ANALYSIS
Dr. Wafaa S. Al wazni
Serological tests for autoimmune disease :
A. Rheumatoid arthritis:
Is a chronic inflammatory disease affecting primarily the joints & periarticular
tissues. Patients with rheumatoid arthritis have abnormal proteins produced in the
synovium of involved joints & circulate in their blood.
These proteins known as rheumatoid factor (RF) which is:
1) A macroglobulin (Ab) with γ globulin activity, their properties are very similar
to those of normal IgM class.
2) RF directed against the Fc protein of IgG.
3) The origin of RF is related to formation of damaged IgG in in infectious joint
disease.
4) RF is anti-antibodies.
Test for RF: Test is designed to detect RF in patient’s serum that reacts with
normal human IgG bound to particulate carriers. Ex:
- SRBCs = passive hem agglutination (Rose-Waaler test)
- Latex = latex agglutination (most popular)
- Bentonite particle = flocculation test.
43
PATHOLOGICAL ANALYSIS
Dr. Wafaa S. Al wazni
B. C - reactive proteins:
A serum globulin reactive against the carbohydrate cell wall of pneumococci
commonly occurs in the serum of patients with rheumatic fever & rheumatoid
arthritis.
This protein usually appears after 14-26 hours of disease beginning & may
increase in concentration by as much as 1000 times its normal amount;
thus it is a useful clinical indicator of disease state. CRP is an  globulin, it has
an (a.a) composition similar to that of Ig but differs from Ig in antigencity.
The elevation of CRP in patient about 0.5 mg/100 ml indicates tissue damage,
inflammation or both.
Test:
a) Precipitation test on slide or tube (serum + anti-CRP).
b) Latex fixation test.
44
PATHOLOGICAL ANALYSIS
Dr. Wafaa S. Al wazni
C. Systemic Lupus erythematous (SLE):
Is an inflammatory disease of connective tissue occurring primarily in women &
characterized by diverse clinical manifestation involving variant organs of body.
The nucleus of PMNs or lymphocyte lyses first & then swells to as much as 3 or 4
times its normal size & then it is ingested by normal PMNs, the latter cell wit the
lysed nucleus as an inclusion body is an LE cell. Abs against LE cell called LE
factor which is an antinuclear antibody that some times react like an Ab.
LE factor (antinuclear factor) found in % 95 of SLE patient & directed against a
large number of tissue components (LE cell).

Test for SLE:
a) Immunofluoresence test:
An antigen (human leukocytes) can be used for determination of antinuclear
factor.
On microscope slide the test serum& fluorescent Ab (anti LE factor) are added
to the Ag; this test called Fluorescent antinuclear antibody test (FANA)
FANA: is the most sensitive screening test for the detection of Abs in the
patients serum directed against various nuclear constituents.
b) Other tests:
Include latex nucleoprotein test & CF test.
45
PATHOLOGICAL ANALYSIS
Dr. Wafaa S. Al wazni
Serological tests for infection disease :
A. Febrile antibody tests
Several diseases cause fever so these diseases known as the febrile diseases
like Salmonellosis brucellosis, rickettsie & tularemia.
Febrile Ags are endotoxin, enzymes or other toxic products, test used for
diagnosis of these Ags known as febrile antibody testes such as:
I) Widal test:
Classic serologic test of Salmonella infection.
O Ag & H Ag prepare from bacteria species used in this test to detect Abs in
patients serum, this test is agglutination test (tube or slide) used widely for
detection of Abs in Typhoid fever, titers greater than 1/160 (O Ag) or
1/80 (H Ag) indicate +ve result. Widal test becomes less useful because of the
cross reacting Abs or previous immunization against typhoid that may
give +ve result with Brucella Ags.
II) Wright agglutination test or Rose Bengal test:
This test is used for diagnosis Brucella, a titer greater than 1/100 (+ve result),
the test is helpful for diagnosis in about the 1 st week of the disease, whereas it
usually takes from 4-6 weeks for the organisms to grow in culture.
III) Tularemia:
The agglutination test is widely used for diagnosis of tularemia which is
caused by Francisella (pasteurella) tulareusis, the organisms is highly
infected & difficult to isolate.
IV) Weil – Felix test:
Agglutination test for Rickettsial disease (typhus fever) which is based on the
fact that certain strains of Proteus vulgaris OX-2, OX-19, and OX-K (non
motile strains) share Ags with Rickettsia spp. So that the Abs against
Rickettsia present in patient serum cause aggl with Ags for Rickettsia spp.
46
PATHOLOGICAL ANALYSIS
Dr. Wafaa S. Al wazni
B. Cold agglutination test:
Are Abs react with human RBCs at (1-10)°C& the agglutination disappear or
dissolve at 25°C or higher; these Abs agglutinate all human RBCs regardless
of blood group& these Abs are found in the serum of patients with primary
atypical Pneumonia after (8-10) days of the disease.
Patient with diseases caused by Adeno virus & mycoplasma Pneumonia have
cold agglutinin; Mycoplasma develop agglutinins for human RBCs type O.
C. Streptococcus MG agglutination test
Patients with primary atypical Pneumonia develop in addition to cold
agglutinins, agglutinins that react with non hemolytic strains of Streptococcus
known as Streptococcus MG. These Abs are specific reacting with capsular
polysaccharide of the organisms, a titer of 1/40 is considered to be infection.
D. Antistreptolysin test (ASOT)
Streptolysin o (SLo) is exoenzyme produce by St. pyogenes which is in
reduced state cause lyses of RBCs & WBCs. It is an antigenic; eliciting the
formation of Abs that effecting neutralizes its hemolytic action, these Abs
appear in serum of patients with Streptococcal infection, therefore the
measurement of serum antistreptolysin O (ASO) is an indicator of
Streptococcal infection (rheumatic fever& glomerulonephritis). ASOT is based
on the fact that ASO can be specifically fixed to streptolysin o in vitro, where
it will neutralize its hemolytic activity.
This test is reported in Todd units, which is the reciprocal of serum dilution
that completely inhibit hemolysis of organism number of RBCs.
250 Todd units (IU)
normal value for adult
330 (IU)
for child
400 (IU)
streptococcal infections
Todd unit: denotes the reciprocal of the highest of test serum at which there
continues to be neutralization of a standard preparation of the streptococcal
enzyme streptolysin o.
Lysis (-ve no Ab)
47
PATHOLOGICAL ANALYSIS
Dr. Wafaa S. Al wazni
ASO (serum Ab) + SLo (Ag)
Ag –Ab + RBCs
No lysis (+ve Ab)
Miscellaneous Serology :
1) Pertussis agglutination test :
The detection of whooping cough can be made with suspensions of bacteria
serotype 1,3 added to equal volumes of serum dilution after incubation for 18 hr
in 37°C, test are read with magnifying glass.
The titer is expressed as a highest dilution yielding complete agglutination.
Complement fixation test is more sensitive for pertussis antibody testing.
2) Leptospiral agglutination test :
Leptospirosis is a group of disease produced by a large number of antigenically
distinct members of genus Leptospira. At least 80 serotypes & sub serotype have
been identified, the disease agent can be detected by agglutination test & hem
agglutination test
(soluble Ag coupled to SRBCs + serum
agglutination)
3) Lance field grouping :
Thermo precipitation test for detection & differentiation the groups of hemolytic
streptococci by precipitin reaction between solution of carbohydrate extracted
from streptococcal cells & antisera prepared by immunizing rabbits with heat
killed suspensions of streptococcal.
Bacterial extract + serum
Incubation
precipitation ring
At 37°C for 10 min
4) Pregnancy testing :
A number of serological tests have been used in pregnancy testing, to detect
human chorionic gonadotropin (HCG) when it appears in the urine during the first
few weeks of pregnancy.
Serological tests are done frequently in the laboratory to detect pregnancy in the
early stages. Laboratory, tests for pregnancy are based on the fact that during
48
PATHOLOGICAL ANALYSIS
Dr. Wafaa S. Al wazni
pregnancy, the placenta produces a hormone called chorionic gonadotropin this
hormone rapidly disappears after delivery.
Human chorionic gonadotropin (HCG) is also produced in other condition as in
choriocarcinoma and in malignant teratomas of the ovaries and tests. In
pregnancy, HCG is produced by the langhans cells of the developing placenta. It
is a glycoprotein with a molecular weight of about 30000.
Immunologic pregnancy tests depends on several factor:
1) The manufacturer's direction must be followed carefully.
2) Collected and delivered of specimen in the laboratory for testing.
3) Stored of antigens.
4) Stage of antigens.
5) Whether the pregnancy normal or abnormal.
6) Presence of interfering substances in the urine (including drugs, proteins, and
red cells).
7) The sensitivity and specificity of the assay procedure and the use of quality
control programs.
 Since detergents may interfere with results, the specimen should be collected in
a disposable urine container if possible. If these are not available, the patients
must be instructed to use a clean rinsed container that dose not contain traces
of detergent or other substrates that might interfere with the test.
 For all HCG test an early morning urine sample is best.
 HCG is lost during storage, so the test should be does as soon as possible.
 The urine should be fresh and clear with specific gravity 1.010.
 The presence of hematuria or proteinuria may cause falsely positive results.
 Phenothiazine anal promethazine drugs may also cause falsely positive results.
49
PATHOLOGICAL ANALYSIS
Dr. Wafaa S. Al wazni
The methods most commonly used now are based on the :
I) Latex particle agglutination inhibition test
This test consists of incubation of the patients urine with anti HCG, followed
by the addition of latex particles coated with HCG, if HCG is present it
neutralizes the Ab so that no agglutination is seen & if no HCG is present in
the urine agglutination occurs between the anti HCG & HCG coated latex
particles. The test gives reliable results about 42 days after the first missed
period.
II) Direct latex agglutination test
The reaction between Ag (HCG in urine) & anti HCG adsorbed on latex
particles, agglutination indicates presence of HCG (+ve).
 Syphilis serology
Causative agent : Treponema palladium
After syphilitic infection two types of Abs appears in the
serum:
a) Non treponemal Abs called reagin which reacts with lipid Ags.
b) Treponemal Abs which react with T. palladium & closely related strains :
1. Ab reacts with protein component of a non pathogenic strain of
T. palladium (Reiter’s strain).
2. Ab reacts directly pathogenic strain of T. palladium (Nichol’s strain).
So that the serological tests for syphilis are of two types, those which detect
reagin Abs against cardiolipin such as VDRL, & those which detect treponemal
Abs include the FTA-Abs & TPI.

Reagin tests for syphilis:
50
PATHOLOGICAL ANALYSIS
Dr. Wafaa S. Al wazni
Infections with T. palladium cause tissue damage of the host, splitting of the
lipoidal fraction which acting as hapten, combines with the protein fraction of the
spirochaete & stimulates reagin production.
Syphilitic regain has two components:
a) Has large M.W bivalent termed reagin which takes part in flocculation test.
b) Is relatively small M.W bivalent termed as coreagin & reacts in complement
fixation test.
Reactions which demonstrate the presence of these reagin & coreagin are
known as standard test of syphilis (S.T.S) which includes these two types:
1. VDRL (Venereal Disease Research Laboratory)
VDRL test can be used quantitatively & qualitatively for detecting reagin in
serum & CSF. This test is replaced by rapid plasma reagin (RPR) test which
include modified VDRL antigen (cardiolipin lecithin coated cholesterol with
choline chloride & also contain charcoal particles) to allow for the testing of
plasma without heating.
Cardiolipin, lecithin coated cholesterol particles + reagin
flocculation
VDRL test can be used qantitively & qualitatively for detecting reagin in
serum & CSF, this test is replaced by rapid plasma reagin (RPR) test which
include modified VDRL antigen (cardiolipin lecithin coated cholesterol with
choline chloride and also contain charcoal particles) to allow for the testing of
plasma without heating.
These Abs also produced by :
 Patients with other infections such as: leprosy, tuberculosis, measles, chicken
pox, hepatitis & infectious mononucleosis.
 Non infectious conditions such as drug addictions & non disease such as
advanced age, pregnancy, recent immunization & in autoimmune disease.
 When positive serological test for syphilis occurs in a patient without syphilis,
it is called biological false positive (BFP) test.
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PATHOLOGICAL ANALYSIS
Dr. Wafaa S. Al wazni
2. Wasser man test
Is complement fixation test used in diagnosis of syphilis, it is required:
a) Ag: cholestrolized alcoholic extract of ox-heart muscle which has slightly
anti complementary activity but with high degree of sensitivity of sera.
b) Human sera: some patients have inhibitors of complement therefore serum
should be heated at 56°C for 30 min.
c) Complements from guinea pig.
d) Indicator system (Anti sheep hemolysin & sensitized SRBCs).
 For quantitative test serial dilution of serum are tested.
The test consists of :
Patient serum + Saline + Ag +Complement
The control consists of :
Serum control + Saline + Ag + Complement
Then the indicator system is added & examine for hemolysis.
Treponemal tests for syphilis :
A. T. pallidium immobilization (TPI) test:
The test is based on the fact that when the organisms incubated with
syphilitic serum & guinea pig complement the organisms lose their motility
(immobilized), examine under dark field microscope.
B. The treponemal hemagglutination (TPHA) test:
The Ag used is a suspension of formalized tanned SRBCs coated with Ag from
Nichol’s strain of T. pallidium & the patient 's serum is absorbed with sorbent
(an extract of a culture of Reiter treponemes) to remove the non specific Abs
52
PATHOLOGICAL ANALYSIS
Dr. Wafaa S. Al wazni
(cross reactive Abs), then the sensitized SRBCs is added, the positive result is
the agglutination of the sensitized SRBCs.
MHA-TP micro hemagglutination test for Abs of T. pallidium (TPHA in micro
titer plates)
ELISA in infection disease
(Enzyme linged immunosorbent assay)
1. Bacterial diseases
- In the assay of antibodies against infection agents.
- To measure the response to Salmonella ‘O’ antigen.
- Application in Cholera serology differentiated ISA, ISG & ISm.
- Serology of Brucella abortus serology of yersinia enterocolitica
- Antibodies to Chlamydiae
- Assay for Klebsiella
- Assay for Legiounairea disease
2. Funcal diseases
- Aspergillus antibody
- Candida albicans antigens
- Aplatoxin B
3. Viral disease
- hepatitis
- cyto megalo virus (CMV) (with newborn & in immuno suppressed patients)
4. Parasitic diseases
- Plasmodium falciparum antigen
- Trypanosomia
- Helminthus
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PATHOLOGICAL ANALYSIS
Dr. Wafaa S. Al wazni
Materials needed:
1. Coating buffer (carbonate-bicarbonate, PH 9.6).
2. PBS Tween
3. Conjugates :
a) Alkaline phosphatase labeled sheep anti human immunoglobulin
b) Horse radish peroxidase labeled rabbit anti human IgG.
4. Substrates :
 For alkaline phosphatase conjugates:- Substrate solution is:
- P-nitrophenyl phosphate (1mg/ml).
- Tablets (5mg) are stored at -20°C in the dark until used.
Immediately before use, one (5mg) tablet is dissolved in each 5ml of %10
diethanol amine buffer at room temperature. It must be used the same day.
Reaction stopping solution -3M NaOH. Read in a spectrophotometer at 405 nm.
 For peroxidase conjugates: substrate solution arthophenylene diamine to be
made up freshly immediately before use.
Of phosphate citrate buffer PH 5 & add 40ml of %30 H2O2.
This substrate is light sensitive & must be used at once.
Reaction stopping solution
25M H2SO4.
Read in spectrophotometer at 492nm.
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PATHOLOGICAL ANALYSIS
Dr. Wafaa S. Al wazni
 Example
 The competition method of ELISA for assaying antigen:
(1)
Adsorb
antibody
to surface
(2.a)
(2.b)
Add enzyme
labelled
antigen
Add labelled
antigen
''un know Ag''
(3.a)
Add enzyme
sub strate
(3.b)
The reference wells containing only enzyme labeled antigen show coloration. The
inhibition of that color change in the wells with test samples is preparation to the
amount of antigen is the test samples.
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PATHOLOGICAL ANALYSIS
Dr. Wafaa S. Al wazni
 Modification of the indirect ELISA for assay of antigen:
Ag
1) Plate coated with antigen
Ab
2) Test sample thought
to contain antigen
mixed together with
reference antigen
No antigen in test sample
Antigen in test sample
3) Enzymes antiglobulin
conjugate added
Anti Ab
Conjugate becomes fixed
to immobilized antibody
4) Enzyme substrate add
Substrate degradation
indicate sample 2
contain no antigen
No substrate degradation
indicate test sample 2
contain no antigen
56
PATHOLOGICAL ANALYSIS
Dr. Wafaa S. Al wazni
The difference in color change between a reference sample containing no antigen
& the test solution in step 2 indicates the amount of antigen in the test samples.
This is competitive assay light antigen concentration results in less color at the
end of the test.
 Solid phase Anti Ab IgM ELISA serology capture :
1) Anti IgM adsorbed to plate
Anti Ab
Ab IgM
2) Test serum sample added
Method A
Method B
Reference enzyme
labelled viral
antigen solution
added
3) Reference viral
antigen solution
4) Reference enzyme
labelled virus specific
antiserum added
5) Enzyme substrate
added
Amount hydrolysed
57 anti IgM
antibody
in sample
PATHOLOGICAL ANALYSIS
Dr. Wafaa S. Al wazni
The color change is proportional to the amount of virus specific. IgM is the serum
tested in step 2.
Fifth\\ Semen Analysis :
Semen analysis is an integral part of the workup of couples consulting for
infertility. The availability of semen renders possible direct examination of male
germ cells, giving precious data that are not accessible for female germ cells.
Semen analysis includes the examination of:
- Spermatozoa
- Other cell present in semen.
- Seminal fluid
All together, these data indications on the testicular function & of the integrity of
the male genital tract.
Type of assay:
1. Descriptive assay
Spermogram
2. Functiinal assay :a. Penetration of cervical mucous (postcoital test, in vitro penetration assay).
b. Binding of sperm to the zona pellucida.
c. Fusion of sperm with zona free hamster oocyte.
d. Hypoasmotic swelling of the flagella.
Macroscopic analysis :
1. PH: The normal PH of semen is alkaline & measured with PH paper.
2. Volume: The volume is related to the number of days since previous
ejaculation & after 3 days of abstinence the range of volume 2-6 ml.
3. Appearance: include smell, color & viscosity.
Microscopic analysis :
1. Sperm concentration.
2. sperm viability (vital staining)
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PATHOLOGICAL ANALYSIS
Dr. Wafaa S. Al wazni
3. spermatozoa motility affect by many factors such as:
a- age of specimen
b- Temperature
4. Morphology analysis
5. Bacteriological analysis
Sixth \\ Biochemical analysis :
Sugar
Determination of Glucose in patient blood may be performed on specimens taken
from patients in a fasting state or on specimen taken 2 hours after the patient has
eaten 100 gm of carbohydrate.
The increased blood glucose concentration, result from an imbalance between
hepatic output of glucose & peripheral outlay of the sugar.
Consequently a hyper glycine is may result from :
1. A normal hepatic output of glucose with a low subversion rate of peripheral
removal.
2. An increase in hepatic production & release of glucose with normal removal
rate by the peripheral tissues.
3. A combine of these factors.
4. Abnormal functioning of hormone may influence glucose levels.
After a period of fasting, the usual amount of glucose in the blood is between 60
& 100 mg/100 ml of blood. The blood glucose level usually increases rapidly
after carbohydrates are ingested but returns to normal in 1.5-2 hours.
 The normal value are different in fast & normal cause which are :
1. Fast 60-110 mg/dl.
2. Random 60-140 mg/dl. (1 dl = 0.1 L is a common metric unit of volume
equal to 0.1 liter or 100 cubic centimeters).
 There are many causes associated with presence of sugar in blood :
1. After large amount of sugar of blood containing sugar are eaten.
2. In cases of acute emotional stress where glucose liberated by liver for
energy
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PATHOLOGICAL ANALYSIS
Dr. Wafaa S. Al wazni
3. It may also be associated with pregnancy, certain type of meningitis,
certain tumors of adrenal medulla & some brain injuries.
 The method used for glucose determination can be divided
into 3 categories:
1. Oxidation methods which depend on the reducing ability of glucose.
2. Aromatic amine methods, which involve a reaction between the aldehyde
group of glucose & the amine group of an aromatic amine.
3. Enzymatic methods which are based on the enzymes glucose, oxidase &
hexokinase.
But the occurrence of sugar in the urine indicates that diabetes mellitus
should be suspected, and test for urinary sugar are commonly used for the
diagnosis and management of this disease. The type of sugar that is present in
the urine in cases of diabetes mellitus is glucose which is also called
dextrose, any condition in which glucose is found in the urine is termed
glycosuria or glucosuria.
Also the glycosuria may caused from another reasons than diabetes
mellitus which are :1. After large amount of sugar or food containing sugar are caten.
2. In cases of acute emotional stress where glucose is liberated by the liver for
energy.
3. It may also be associated with pregnancy, certain type of meningitis,
hypothyroidism, certain tumors of adrenal medulla and some brain injuries.
Virtually all tests for urinary sugar may be classified as one of two
types:1. Nonspecific test for sugars in general, which are based on the ability of glucose
to act as a reducing substance.
2. Specific tests for glucose, which are based on the use of the enzyme glucose
oxidase.
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PATHOLOGICAL ANALYSIS
Dr. Wafaa S. Al wazni
The tests that are based on the reducing ability of glucose are not specific for
glucose. In these tests, the glucose is merely acting as a reducing agent, and any
compound with a free aldehyde or ketone group will give the same reaction.
All these substances and glucose have the ability to reduce a heavy metal
from a higher to lower oxidation state. Usually copper II ions are reduced to
copper I ions.
Metabolism of Carbohydrates
The polysaccharide (starch) degradation in mouth into maltose by the action of
salivary amylase, a pancreatic amylase in the duodenum continues the hydrolysis
or breakdown of starch and glycogen into maltose (disaccharide).
Monosaccharide which obtained by degradation of disaccharide absorbed through
the wall of small intestine into the blood, the major portion going into the portal
circulation and to the liver. Insulin is responsible for maintaining a healthy level
of glucose in the blood.
Glucose can also be provided to the blood by the process called gluconeogenesis,
the production of glucose from fat and protein, this takes place in the liver, where
glucose is derived from certain amino acids (protein) and glycerol (from fat) by
glucocorticoid hormones produced by the adrenal cortex decreased blood glucose
and decreased glycogen storage in cell stimulate this process.
After a period of fasting, the usual amount of glucose in the blood is between 60
and 100 mg/100 ml of blood, depending on the method of analysis, the blood
glucose level usually increases rapidly after carbohydrates are ingested, but
returns to normal in 1-2 hours.
The blood glucose concentration above which glucose is excreted in the urine is
called renal threshold. For most individual the renal threshold for glucose is
160-170 mg/100 ml.
Several hormones have the affect of raising the blood glucose concentration. The
actions of growth hormone and adrenocorticotropic hormone (ACTH), which are
secreted by the anterior pituitary, are opposite (antagonistic) to that of insulin and
tend to raise the blood glucose level. Hydrocortisone and other steroid produced
by the adrenal cortex stimulate gluconeogenesis, the production of glucose from
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PATHOLOGICAL ANALYSIS
Dr. Wafaa S. Al wazni
fat and protein epinephrine, which is secreted by the adrenal medulla, stimulates
glycogenolysis, the conversion of glycogen to glucose. Thyroxin which is
secreted by the thyroid, also stimulates glycogenolysis and contributes to an
increased blood glucose level, besides increasing the rate of absorption of glucose
from the intestine.
 Liver Function:
Normal liver function
The liver responsible for many metabolic, storage, excretory and detoxifying
function. More specifically, the liver is a major factor in the metabolism of
carbohydrates, lipids and proteins, in both intermediary metabolism and the
synthesis of many essential compounds. Many necessary enzymes and coenzymes
for carbohydrate, lipid and protein metabolism are present only n cells of the
liver. Glycogen is formed, stored and converted back to glucose in the liver.
Energy derived from food is made available to the cells of the body through the
process of glycolysis of the high – energy bonds in adenosine triphosphate (ATP),
which are formed by oxidative phosphorglation in the cells of the liver.
The liver is essential sin the formation and secretion of bile, bile pigments and bie
salts, which are necessary for digestion.
These substances are derived from bilirubin, a major by – product of the
destruction of red blood cells. In addition, the liver is the site of formation and
synthesis of many of the factors involved in the clotting of blood.
These important functions of the liver may be altered when the liver is diseased or
damaged. Numerous laboratory teats are available to determine both the existence
of the liver disease and the extent, location and type of damage so that appropriate
treatment can be initiated. There is not one test will give a complete clinical view
of liver function. One test of liver function I for the presence and concentration of
bilirubin in the blood and the urine.
1) Bilirubin metabolism
Bilirubinis a normal product resulting from the breakdown of red blood cells.
As part red blood cell degradation, the heme portion of hemoglobin molecule is
converted to the bile pigment Bilirubin by the Reticulo Endothelial (Re) system,
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PATHOLOGICAL ANALYSIS
Dr. Wafaa S. Al wazni
primarily by RE cells in the liver, soleen and bone marrow. A total of
approximately 6 gm of Hb is released each day as overage RBCs are eliminated
from the body. The cells of the RE system first phagocylizes the RBCs, then
convert the released Hb through a complex series of reactions in which the heme
portion of the molecule is finally converted to a vivid yellow pigment (Bilirubin).
An increase in the concentration of bilirubin in RE cells, bilirubin is not soluble in
water. Therefore it is transport from the RE cells through the blood to the liver
cells as a bilirubin – albumin complex. This water – insoluble form of bilirubin is
often refferd to as free bilirubin or unconjugated bilirubin.
When free bilirubin reaches the liver, it is converted to a water – soluble product
by the kupffer cells of the liver. It is made soluble by conjugation with glucuronic
acid and some other hydrophilic substances to form bilirubin glucuronide.
Water – soluble Bilirubin, often referred to as conjugated bilirubin, can be
eliminated from the body by way of the kidney or the intestine. Normally,
conjugated bilirubin is excreted by the liver into the bile, transported to the
common bile duct and then to gallbladder. Where it is concentrated and emptied
into the small intestine.
In which most of the bilirubin is convert to urobilinogen by the action of certain
bacteria. Urobilinogen in the intestine is either eliminated from the body
unchanged or oxidized to the coloured compound urobilin .
Abnormal bilirubin metabolism (Jaundice)
Jaundice is a condition that occurs when the serum bilirubin concentration
becomes grater than normal and there is an abnormal accumulation of bilirubin in
the body tissues. Since bilirubin is a vivid yellow pigment an accumulation in the
tissues results in a yellow pigmentation of the skin, the sclera and the mucous
membranes. The classification of the jaundices causes are:
a) Prehphtic jaundice (hemolytic jaundice)
It occurs in conditions in which there is increased destruction of RBCs this type
of jaundice is found in infants with:
1) Blood group incompatibilities.
63
PATHOLOGICAL ANALYSIS
2) In neonatal physiological jaundice
3) Increased production of free bilirubin.
Dr. Wafaa S. Al wazni
There is an increased formation of conjugated bilirubin and a subsequent
increased formation of uvobilinogen. So that the increased of uvoblinogen in stool
and the excess goes to circulation and then excrete with urine.
b) Hepatic Jundice
Results from condition that involve, the liver cells directly and prevent normal
excretion such a condition might be specific damage such as conjugation failure
in neonatal physiological jaundice, where there is an enzyme deficiency. Disease
of conjugation failure result in an increased concentration of unconjugated
bilirubin in the blood. Diffuse or overall hepatic cell involvement occurs in such
condition as viral hepatitis, toxic hepatitis caused by heavy metal or dray
poisoning and cirrhosis. In these cases the ability of the lives cells to remove and
conjugate free bilirubin is decreased resulting in increased amounts of free
bilirubin in the blood.
c) Post hepatic jaundice
Is also referred to as obstructive jaundice. It occurs when the common bile duct is
obstructed by stones, tamers, spasms or a stricture. As a result the bilirubin that
has been conjugated by the liver is returned back into the liver sinusoids and the
blood.
Clinical significance of bilirubin
An increased serum bilirubin concentration may indicate either:1. Increased destruction (hemolysis ) of RBCs.
2. Impaired excretory function of liver cells.
3. Obstruction of the bile flow.
In obstruction jaundice there is an increased in total bilirubin, and in direct
bilirubin, while in hemolytic jaundice there is an increase In total bilirubin and
direct fraction because the increasing occur in free bilirubin with liver damage
such as viral hepatitis, both the free and conjugated bilirubin increase and total
direct and indirect fraction are elevated.
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PATHOLOGICAL ANALYSIS
Dr. Wafaa S. Al wazni
Elevations in serum bilirubin occur in many infant in the first few days of live.
This is especially true of premature infants such neonatal physiological jaundice
may involve either a deficiency of the enzyme that transfers glucuronate group
onto bilirubin or liver immaturity. Bilirubin can not be conjugated and the
concentration of free or indirect bilirubin is increased in blood.
2) Protein
Although plasma protein alternations are not usually specific for a particular
disease condition but any observation revealing an abnormality in the plasma
protein indicate that some pathologic or physiologic condition & a particular
significance in respect to the internal metabolism of the protein is the functional
state of liver & kidney.
The hypoproteinemia is most associated with a lack of proper diet or poor
absorption of dietary constituent from the intestinal tract or loss of the protein
result from burns, draining wounds & disease or liver disease & in some cause the
decrease in total protein may be associated with pregnancy & lactation.
The hyperproteinemia occurs with less frequency & is most associated with
shock, dehydration & lympho sarcoma or plasma cytoma.
The total serum or plasma proteins can be determined by 2 methods:
1. Refractometric methods
2. Biuret methods
 The normal value for protein is range from 6.2-8.2 G/dl.
3) Blood uric acid
The determination of blood uric acid as a liver function test. The blood uric acid
levels were elevated signification in hepatocellular Jaundice but were normal in
obstructive & hemolytic Jaundice, because the site of conversion of uric acid to
allantion by Uricase in the liver.
Normal blood values for uric acid in the human are between 3-7 mg/dl & the
increase above 7 mg/dl may be an indication of liver disease & may have liver
damage.
Then
65
to
PATHOLOGICAL ANALYSIS
Purins
uric acid
Dr. Wafaa S. Al wazni
allantion: in liver
4) Serum Enzymes activity
Alteration in serum enzyme activity due to malfunctioning of the liver occurs
as a result of three processes:
1. Elevation of enzymes due to disruption of hepatic cells as a result of necrosis
or altered membrane permeability.
2. A decrease in concentration in the serum resulting from impaired synthesis by
the liver.
3. Elevation in enzymes levels due to the lack of biliary excretion.
 The Gpt (Glutamic pyrunic transaminase) is present in large quantities in
the liver & it is increased in serum when cellular degeneration or destruction
occur in these organs; but the Got (Glutamic oxulacetic transaminase)
it appears in extremely high concentration in muscles both skeletal & cardiac
& the serum Got level may be increased with liver disease, therefore can not be
considered to be specific test for liver damage but Gpt considered specific test
for liver damage & diseases.
 The 2 enzymes present in small quantities in serum :
a) The Got= 40 unit/ml
b) The Gpt = 45 unit/ml
As a consequence, normal tissue destruction & subsequent enzyme release which
lose their principal function within the cell. The increases observed in serum
reflection of cellular destruction or disease.
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PATHOLOGICAL ANALYSIS
Dr. Wafaa S. Al wazni
 Kidney function:
The kidneys are the chief organs of regulation of the internal environment of the
body. This occurs by this works:
1. Limination of water introduced into the body in excess of the amount required
for normal metabolic processes.
2. Elimination of inorganic element according to the needs of the body.
3. Elimination of non volatile end products of metabolic activity.
4. Elimination of certain foreign toxic substances.
5. Formation & excretion of substances such as hydrogen & ammonia.
Analysis of urine (end product of kidney function) often reveals alteration typical
of disease of that organ but in addition may provide information concerning
alteration in other physiological processes in the body.
 Gross examination to urine:
1. Urine volume
2. Color
3. Specific gravity
4. Transparency
 Chemical examination:
Although the urine contains a large number of organic substances, only a few
of these are of clinical significance in urine analysis.
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PATHOLOGICAL ANALYSIS
Dr. Wafaa S. Al wazni
Analysis for chemical composition of urine routinely
includes the following tests:
1. Reaction (PH):
The normal hydrogen ion concentration of urine is dependent upon the type of
diet. Man with vegetable diet has a tendency to produce alkaline urine; where as
acid urine is normal in man with diet that has high protein content.
The hydrogen ion concentration of urine can be readily determined by the use of
litmus paper.
2. Protein:
The detection of protein in the urine is always considered pathologic & any
persistent protein urea should be investigated, because the normal urine does not
contain detection protein & most of the protein that passes the glomerular filter is
reabsorbed in the tubules, but when the glomerular capsule is damaged, protein
molecules can pass through & end up in the urine.
There is a correlation between the presence of casts in the urinary sediment &
protein urea since casts are made of precipitated protein. The presence of casts
with protein urea distinguished as an upper urinary tract (kidney) disorder.
The presence of WBC & bacteria & protein in the urinary sedimentation, this
distinguish the lower urinary tract without renal involve.
 A wide variety of methods for the detection of protein in the urine are available
to the laboratory most of which dependent on available kit.
One of the methods use strong acid such as that incorporated in Robert’s
reagent which is consistent of:
 1 part of concentrated uric acid
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PATHOLOGICAL ANALYSIS
Dr. Wafaa S. Al wazni
 Saturated solution of magnesium sulfate prepared by :
- 770 gm magnesium sulfate
- 1000 ml D.W
 The result represent while ring of the Junction after 2-3 minute.
3. Glucose:
The occurrence of sugar in the urine indicate that diabetes mellitus (D.M) & the
condition in which glucose is found in the urine is termed glucose urea, this occur
as a result of fear, excitement & restraint. This appearance of glucose in urine is
thought to be the result of hyper glycemia that occurs to an increased search of
epinephrine which leads to a rapid mobilization of glucose & glucosuria occurs
after heavy meal of carbohydrate.
 The normal value for protein in urine range from 1-2 G/d.
 A large number of methods are available for both qualitative & quantitative
measure of glucose in the urine & all of them depending on available kit.
4. Blood urea:
The levels of urea in the blood are affected not only by alterations in renal
function, but maybe altered by certain physiological factors or diseases nor
primarily of renal origin. Physiologically blood urea nitrogen level is increased
with dietary increase in protein. Any factor that decreases glomerular filtration
rate will increase blood urea nitrogen.
 The normal value of blood urea range from 20-45 mg /dl.
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