UNIT-IV.

CLINICAL PATHOLOGY

1. SPUTUM EXAMINATION:
Definition: Sputum is a thick fluid in the lungs and in the adjacent airways. it is a mixture of
saliva and mucus coughed up from the lungs. it also consist of a variety of cellular and non
cellular substances.

COLLECTION OF SPUTUM:
1. PREPARE
Brush your teeth and rinse your mouth with water. You can also try breathing deeply over a
pan of boiling water.

2. COUGH
Take a deep breath, hold it for five seconds, and then slowly breathe out. Repeat this three
times. Then, cough hard until you produce sputum.

3. COLLECT
Spit the sputum into a sterile container. You should aim to collect at least 1–2 teaspoons, or
5 ml, of sputum.

4. LABEL
Write the date and your name on the container.

5. STORE
Keep the sputum in the refrigerator until you can submit it to a laboratory. You should
transport it the same day you collect it, unless instructed otherwise.

IDEAL SAMPLE:
1. early morning
2. free of contamination by food particles
3. deep specimen which is thick and not a sample of saliva
4. Minimum 3-5 ml in quantity.
 PRESERVATION AND TRANSPORT:
1. STORE

Keep sputum in a refrigerator at 4°C to prevent the growth of unwanted microorganisms. Do

not store it for more than one week.

2. TRANSPORT

Place the sputum in a leak-proof biohazard bag with a sealed lid and absorbent
material. Transport it in a cooler with chilled ice packs as soon as possible after collection.

3. PACKAGE

Use a wide-mouthed, unbreakable, leak proof, screw-capped container with a volume

capacity of 50 ml. Make sure the container is translucent so you can observe the specimen
volume and quality without opening it.

4. LABEL

Label the container with the patient's name and identification number, collection date, time,
and location.

5. PROTECT

Protect the specimen from light

6. Preservation sputum sample with cetylpyridinium chloride( CPC) for tuberculosis cultures.

METHOD OF SPUTUM EXAMINATION:

mainly three methods used in Sputum examination.

1. Physical Examination
2. Chemical Examination
3. Microscopic Examination.

1. Physical Examination Of Sputum:

1. Quantity:
Large amount of purulent Sputum- Bronchiectasis. Lung abscess
Large amount of watery Sputum with pinkish tinge – Pulmonary oedema ( fluid collection in
the Lungs)
2. Colour:
colour of the Sputum is Indicates type of infection of inflammation.

White or Clear - Normal

Yellow or dark Yellow – indicates a viral or Bacterial infection.
Green – indicates a infection by Pseudomonas
Blood- indicates carcinoma, tuberculosis

3. Odur(smell):
Foul-smelling sputum can indicate anaerobic infections. foul smelling sputum is observed in
bronchiectasis and lung abscess.
4. Consistency:
The consistency of sputum can indicate the type of infection

Mucopurulent- Commonly found in bacterial pneumonia or bronchitis

Scanty watery- Often found in atypical pneumonia
 2. CSF EXAMINATION:

Definition: Cerebrospinal fluid is a clear, colourless fluid found around the brain and spinal
cord.
It is produced in the brain ventricles by the chorid plexus and epydymal cells and function
of CSF is cushions , protect and delivers nutrients to the brain and spinal cord.

Collection of CSF:
The specimen should be collected by a physician.
Three method are to be used to collection of CSF
1. lumber puncture or spinal tap
2. ventricular puncture
3. cisternal puncture

1. lumber puncture:
 The patient lies on their side or sits on an exam table.
 The patient's back is cleaned and an anaesthetic is injected into the skin.
 A thin, hollow needle is inserted between two vertebrae in the lower spine.
 A small amount of CSF is withdrawn for testing.

2. ventricular puncture:
A hole is drilled in the skull and a needle is inserted directly into one of the brain's
ventricles. This method may be recommended for people with possible brain herniation .

3. Cisternal puncture:
A needle is placed below the occipital bone (back of the skull). This method is dangerous
because it's close to the brain stem

the fluid is collected into three sterile test tubes, about 2ml in each tube.

first tube for biochemistry studies

second tube for microbiological examination
third tube for cell and cell type is pathology section and also additional collection may be
required if examination for malignant cells is required( cytology section.)

Function of CSF:

 Protection: CSF acts as a shock absorber to protect the brain and spinal cord from injury
caused by sudden movement or impact. It also helps maintain the brain's shape by reducing
the downward force on the brain.
 Buoyancy: CSF reduces the brain's effective weight from around 1,500 grams to about 50
grams. This reduces the force applied to the brain and cerebral vessels during injury.
 Waste removal: CSF helps clear waste products from the central nervous system.
 Homeostasis: CSF helps maintain the homeostasis of the brain's interstitial fluid.
 Intracranial pressure regulation: CSF regulates intracranial pressure. The production of
CSF can be increased or decreased to meet the central nervous system's needs.

Preservation and Transport of CSF:

Preservation of CSF:
Specimens for culture should not be refrigerated or exposed to extreme cold, excessive heat,
or sunlight.

Transport of CSF:
CSF specimens should be transported to a microbiology laboratory as soon as possible
They should be transported at temperatures between 20°C and 35°C.

Normal Composition of CSF:

colour- colourless
appearance- clear
no clot formation of standing
specific gravity:1.003-1.008
protein:15-45mg/dl
glucose: 40-80mg/dl
chlorides: 700-750 mg/dl

CSF EXAMINATION:
mainly three methods used in CSF Examination
 Physical Examination
 Chemical Examination
 Microscopy Examination.

Physical Examination:
Colour:
colourless – Normal
Red colour-traumatic tap
yellow- increase in billirubin , jaundice
pink/orange- blood breakdown product
Green- infection.
Appearance:
Clear- normal
t-urbid – pus or RBC presentcloudy- presence of leukocyte.
Cobweb:
cobweb coagulum form on the standing in case of tuberculosis meningitis.

Chemical Examination:
various method and technique used to conduct the chemical examination of CSF for
determine the volume of protein, glucose, chlorides and value of specific gravity of CSF.
total protein test:
normal value of protein of CSF range in 15-45mg/dl. the reducing the protein in CSF is
indicates meningitis.

total glucose test:

normal value of glucose of CSF is 40-80mg/dl . the CSF glucose is about 60% of
plasma values. it is necessary to collect a random blood sugar sample at the same time as the
lumbar puncture.

total chloride test:

normal value of chloride of CSF is 700-750mg/dl. the reducing chloride in CSF is
indicates tuberculous meningitis.

Microscopic Examination of CSF:

in clinical pathology section was used microscopic examination of CSF for cell count and cell
type(differential count)

Cell Count(leucocyte count):

 mix the CSF sample carefully
 fill the neubaur chamber with the CSF sample
 if CSF appears clear, use it undiluted
 if CSF appears cloudy, make 1:20 dilution by using WBC ouoette
 leave the counting chamber on the counter for 5 minutes to allow the cells to settle
 place the chamber on the microscope stage.
 count the cells in the 4 corner square
 if the count is low , count all the nine square

basic formula for cell count

number of cell counted x dilution x depth
Number cells/cumm= --------------------------------------------------
area counted
calculation:
number of cell/cumm= total number of cells x 50

Cell type (differential count):

the specimen should be centrifuged, centrifugation at 1000 x g for 10-15 min. the supernatant
should be drawn off with a Pasteur pipette. one or two drops of sediment should be used to
prepare a smear.
the smear is stained with Leishman stain similar to a peripheral smear
if facilities are available, cyto centrifugation instrument can be used
the cells are counted under high power
in adults, normal CSF contains small number of lymphocytes and monocytes upto 7%
nutrophils are normal.

3. EXAMINATION OF BODY FLUIDS.

The commonly examined body fluids in the laboratory include Pleural, Pericardial
and Peritoneal fluids.
The body fluids are serous fluids which are plasma ultrafiltrates. Pleural fluids surround the
lungs, Pericardial fluids is around the heart and Peritoneal fluid is around the abdominal
and pelvic cavities.

It serves as lubricant to prevent friction between the two layers of the covering
membranes.

Normally a small amount of fluid is present within these body cavities. Increase in the
volume of the fluid in these cavities is known as “ Effusion”

Examination will help to differentiate a transudate from exudates, causes of which

are different.

Collection of Body Fluids:

collection of fluid procedure is called a Tap. it is an invasive procedure to remove

fluid from the space for diagnostic purpose.
inject the aneshesia to patient. and used to Hollow needle for extract the body fluids from
human body.
Pleural fluid:
Collected by thoracocentesis, which involves inserting a needle through the body wall
into the pleural space, the area between the lungs and the chest wall.

Pericardial fluid:
Collected by pericardiocentesis, which involves inserting a needle through the body
wall into the pericardium, the sac around the heart.

Peritoneal fluid:
Collected by paracentesis, which involves inserting a needle through the body wall
into the peritoneal cavity. Peritoneal washing samples can also be collected by instilling
saline into the peritoneal cavity and then withdrawing the fluid.

Preservation and Transport of Body fluids:

Pleural fluid:
Transport in a collection container and bag to prevent leakage or breakage.
Refrigerate for up to two days, and call the laboratory if it's older than that.
Pericardial fluid:
Transport as soon as possible, as freshly tapped samples are best for cytological
examination. If you can't process it immediately, refrigerate at 4°C and transport on ice or in
a cool box. You can store it at room temperature for up to seven days, or in the refrigerator
for up to seven days. Freezing it will preserve it for up to 28 days.
Peritoneal fluid:
Collect in a leak-proof container and transport in a sealed plastic bag. Large volumes
or whole dialysate bags may require special transportation.

Body Fluids Examination:

Physical Examination:
Physical Examination of the body fluids is Can detect abnormal fluid accumulation.

Chemical Examination:
Chemical examination of body fluids is include pH, glucose, lactate dehydrogenase
(LD), and total protein.

Protein Estimation:
This helps to differentiate transudate from exudates
Glucose Estimation:
low glucose in the body fluids usually indicates bacterial infection.

Microscopic Examination:
Cell Count(leucocyte count):
 mix the sample carefully
 fill the neubaur chamber with the CSF sample
 if appears clear, use it undiluted
 if appears cloudy, make 1:20 dilution by using WBC ouoette
 leave the counting chamber on the counter for 5 minutes to allow the cells to settle
 place the chamber on the microscope stage.
 count the cells in the 4 corner square
 if the count is low , count all the nine square
basic formula for cell count
number of cell counted x dilution x depth
Number cells/cumm= --------------------------------------------------
area counted
calculation:
number of cell/cumm= total number of cells x 50

Cell type (differential count):

the specimen should be centrifuged, centrifugation at 1000 x g for 10-15 min. the supernatant
should be drawn off with a Pasteur pipette. one or two drops of sediment should be used to
prepare a smear.
the smear is stained with Leishman stain similar to a peripheral smear
if facilities are available, cyto centrifugation instrument can be used
the cells are counted under high power

Stains used for body fluids analysis:

Gram stain- for bacteria.
Ziehl-Neelsen stain- for acid fast bacteria.
Difference between Transudate and Exudates:
Exudate Transudate
Filtration of plasma due to increased
Due to increased vascular permeability hydrostatic pressure, normal vascular
permeability
Cloudy/purulent Clear, straw colours
Specific gravity high Specific gravity low
Protein high Protein low
Cell count high Cell count low
Types of cells- increased lymphocyte and Types of cells- few lymphocyte and
neutrophils mesothelial
Bacteria present Bacteria Absent

4. SEMEN ANALYSIS:
it is a seminal fluids consist of spermatozoa or sperms suspended fluids called “Semen”.
or
it is the organic fluid ejaculated from the male reproductive tract which contains sperm cells.
It also contains liquid to form a seminal plasma which helps to keep sperm viable.

Composition of Semen:
The semen fluid is mainly generated from the secretions of male reproductive organs.
Semen consists of potassium, enzymes, citric acid, phosphorylcholine, free amino acids,
fructose, prostaglandin, and zinc. 46% to 80%of the fluid is given out by the seminal vesicles,
13% to 33% by the prostate gland, 5% from the testicles and epididymis, and 2% to 5% from
Bulbourethral and urethral glands.

Indication of Semen Analysis:

 In case of infertility
 success of vasectomy
 Medicolegal- in rape cases
in clinical pathology, the first two are important.
Collection , Preservation and Transport of Semen:
 Abstain from sexual activity: Avoid sexual activity for 2–5 days before collecting
the sample.

 Pick up a container: collecting the sample at home, you can pick up a clean
container from the lab.

 Collect the sample: masturbate into a sterile container or cup,

 Return the sample: return the sample within one hour of collection. Store the sample
at room temperature during transport and do not freeze it.

 Identification: In the container must contain the following information:

Patient Name
Date
Sampling time
Abstinent days
 during the transportation of the specimen, it should not be exposed to extremes of
temperature.
 during transport to the laboratory, the sample should be kept between 200c -370c
  in delay after collection till submission to the laboratory be more than one hour. the
sample can not be preserved.

Examination of Semen:
Physical Examination :
Volume: The normal volume of semen is 2–6 mL after 2–7 days of sexual abstinence.

Colour: normally semen has an opaque grey colour

pH: The normal pH range is 7.2–8.2. An acidic pH may indicate a blocked seminal vesicle,

while a basic pH may indicate an infection.

Sperm count: The normal total sperm number is 39 million or more per ejaculate.

Sperm morphology: More than 4% of sperm should be normal forms.

Sperm vitality: More than 58% of sperm should be live.

Viscosity: This measures the semen's resistance to flow. fresh semen has high viscosity.

Liquefaction: Semen normally coagulates upon ejaculation and liquefies within 15–20

minutes.

Chemical Examination:

 Fructose test: to determine the amount of energy available to the sperms for moving.
the normal seminal fructose level is 150-300mg/dl. it is measured by qualitatively by
Resorcinal test.

 Acid Phosphate test: to determine acid phosphate in semen.

 Barberio’s test: to detect the spermine in semen.

Microscopic Examination :

microscopic examination semen also known as Semen analysis it include Sperm count,

Motility, morphology, Abonormalities and Other cells.

Sperm Count:

Procedure:
  After liquefaction has taken place, gently mix the specimen
 draw semen to the 0.5 mark of a WBC pipette and draw in special semen diluting
fluid to the 11 mark and mix well
 Load the Neubauer Chamber
 Allow the sperms to settle
 count in the 4 corner squares, as WBC count
 the formula for calculation is similar to the WBC formula
 the normal sperm count is between 15-45 millions/ejaculate
low sperm count is called- Oligospermia
absence of sperms is called Azoospermia.

Motility:

Procedure:

 mix the samen sample well

 remove a small quantity of semen immediately after mixing
 prepare a wet preparation by placing a drop of semen onto a clean glass slide. cover it
with a square cover slip.
 take care to avoid the formation and trapping of air bubbles between the cover slip
and the slide.
 wait for about a minute for the sample to stop drifting
 asses approximately 200 sperms for the percentage of different motile categories.

Grading of Motility:
 Progressive Motility- spermatozoa moving actively, either linearly or in a large
circle, regardless of speed.
 Non- progressive Motility- motility with an absence of progression
 Immotile- no movement
the lower limit for total motility( progressive & non progressive) is 40%.
the lower limit progressive motility is 32%

Morphology:
procedure:
 after liquefaction make a thin smear on a slide similar to blood smear
 let it air dry
  the smear can be stained with Leishman stain as for blood smear
 the smears are studied with light microscope
the normal spematozoan is 50-70 micron in length, with a large oval head, a small neck and a
long slender tail, the last taking up about 90% of total length.

Abnormalities of morphology:

Head defects: The head may be too large or small, misshapen, or have multiple heads

Mid piece defects: The mid piece may lack mitochondria, which are essential for sperm
motility and energy production

Tail defects: The tail may be coiled, stump-like, or have multiple tails
 abnormal shaped head
 abnormally sized head giant or minute
 double head
 irregular distribution of the chromatin
 neck may be absent, swollen
 tail may be doubled, rudimentary or absent.
Other cells: during the examination of semen for sperm motility and morphology the
presence of other cell should be noted such as red blood cells, neutrophils, epithelial cells,
immature cells from the testis as well as various crystals.

Diagram of Sperm :
 SPERM

V. CYTOPATHOLGY
Cytology, also known as cytopathology, is the study of cells and their structure, function, and
pathology.

Method of Cytopathology:
classified the cytopathology into two types
 Expoliative Cytology
 Intervative cytology

Expoliative Cytology:
the study of cells by naturally collecting a sample from human body and it is not needed to
any special equipment and instruments.
ex: urine, sputum, semen, etc.

Intervative Cytology:
the study of cells , sample was draw from human body by using a Fine Needle Aspiration.
ex: CSF, body fluids, blood ,etc