Module 3 - Clinical Pathology Sujoy Tontubay.pptx

Module 3 – Clinical Pathology
By
Sujoy Tontubay (B.Sc. M.Sc. – Biomedical Laboratory Science, Post Graduate Diploma in Epidemiology and Public health)
Assistant Prof. Clinical Diagnosis – Jain University of Allied Health Science, Bangalore.
Former Assistant Professor in Allied Health - Medical Laboratory Technology, iLEAD. Former Guest Lecturer in GNIPST, Lokenath
Educational Institute and Dumdum Institute of Health care education, Examiner at MAKAUT.

Introduction to Clinical Pathology
Clinical pathology is the branch of Laboratory medicine that deals with
the diagnosis and management of diseases based on the laboratory
analysis of bodily fluids, such as blood, urine, and cerebrospinal fluid.
Clinical pathology encompasses various disciplines, such as hematology,
coagulation, immunology, clinical chemistry, microbiology, and
transfusion medicine.

Why Blood collection is important?
• Most of the samples that are collected for different types of
biochemical, pathological, hematological and
microbiological test is the blood. That’s why drawing blood
is the most important procedure for any laboratory
professionals or technologist or clinician.
• The collection of blood is done for many reasons. To do
research in the field of medical sciences, for saving lives in
hospitals and blood banks.
• Blood collection used an all labs to find out why symptoms
are appear to the patients.

Becoming A Phlebotomist ?
A Phlebotomist is a person responsible for drawing blood
from patients for lab tests, transfusions, or donation.

Patient Positioning
• Outpatients(OPD) : Sitting On A Chair.
• Inpatients(IPD) : Lying On Bed Or Sitting On A Chair.
• Hand position: When collecting blood from a hand vein, it is best practice to
position the patient's hand slightly downward with the top of the hand facing you.
The fingers of the patient's hand should be rolled underneath, forming a loose fist.

Reagents and Equipment's needed for blood collection:
• Syringes
• Cotton
• Tourniquet
• 70% Alcohol
• Gloves
• Vacutainer (Tube/Vail)

Vein Selection
• Choose the veins that are large and accessible.
• Large veins that are not well anchored in tissue frequently
roll, so if you choose one, be sure to secure it with thumb .
• Avoid bruised and scarred areas.

Veins Used For Drawing Blood
• Median Cubital Vein – First Choice, Well Supported.
• Cephalic Vein – Second Choice.
• Basilic Vein- Third Choice, Often The Most Prominent Vein, But It
Trends To Roll Easily(unstable) And Makes Venepuncture Difficult.

Tourniquet Application
• Apply approximately 3-5 inches
above antecubital fossa.
• Don’t apply too much tight or
too much loose.

Palpate
• After the tourniquet
apply , patient have to
clench first.(close or hold
hand tightly). Then feel
the vein using your index
finger.

Cleansing The Site
• After selecting vein, clean the puncture site with a cotton ball
saturated with 70% alcohol. Then rub the alcohol swab in a circular
motion moving outward from inward site.
• After cleaning the site, do not touch the site at the time of collection.

Performing The Venipuncture :-
• Clean the selected area with spirit(70% alcohol) swab.
• The patient arm is gripped tightly with the help of tourniquet.
• The vein is penetrated by positioning the needle at 20 to 30 degree angle.
And the syringe hole must be upward position. then draw the blood by the
vacuum pump.
• After blood has been drawn, the patient gripping hand should release the
first and the tourniquet is also released. And then release the syringe slowly.
• A cotton ball is held firmly over the venepuncture site as soon as the needle
is removed.
• After removing the needle the collected blood is dispensed in the
appropriate tubes. (vacutainer tube /vail)
• The needle should be disposed by using needle destroyer or throughout into
a non penetrable containers.

After the blood is being collected, blood will be transferred
to certain vials for different types of tests—discussed in the
next slides. These are all anticoagulated vials except the red
one. Red vial is also called clot vial because there is no
anticoagulant present in this vial. Whole blood is collected
in blood collection tubes or syringes that contain the
appropriate anticoagulant to inhibit coagulation.

• Red Top
• ADDITIVE : None
• MODE OF ACTION : Blood
clots, and the serum is
separated by centrifugation
• USES : Chemistries,
Immunology and Serology,
Blood Bank (Crossmatch)

• Purple Top
• ADDITIVE : EDTA
• MODE OF ACTION : Forms
calcium salts to remove calcium
• USES : Hematology (CBC) and
Blood Bank (Crossmatch);
requires full draw - invert 8
times to prevent clotting and
platelet clumping

• Light Blue Top
• ADDITIVE : Sodium citrate
• MODE OF ACTION : Forms
calcium salts to remove
calcium
• USES : Coagulation tests
(protime and prothrombin
time), full draw required

• Light Gray Top
• ADDITIVE : Sodium fluoride and
potassium oxalate
• MODE OF ACTION : Antiglycolytic
agent preserves glucose up to 5 days
• USES : Glucoses, requires full draw
(may cause hemolysis if short draw)

• Green Top
• ADDITIVE : Sodium heparin or
lithium heparin
• MODE OF ACTION : Inactivates
thrombin and thromboplastin
• USES : For lithium level, use
sodium heparin For ammonia
level, use sodium or lithium
heparin

Storage of Blood Sample
• Whole blood is usually kept at 4 degree C for a few
days.
• In order to ensure the integrity of the blood samples
for a longer period of time, it is recommended to
freeze them at -20 degree C.
• To keep samples for several years, other requirements
are necessary, such as freezing at - 80 degree C.
• The sample must be processed as early as possible. If
delay is inevitable, the serum must be separated and
refrigerated as this delays chemical changes. The
concentration of most of the commonly determined
constituents remains stable up to a week if kept at 4
degree C and longer if frozen. Some enzymes are,
however, unstable at 4 degree C, and should be frozen
as soon as possible.

Transport of Blood Sample
Specimen should be delivered as soon as possible to the laboratory,
usually within 2 hours but in some cases they should be sent
immediately. Passage of time affects concentration of many blood
constituents once it has been shed from the body.

Serum Preparation –
Collect whole blood in a microcentrifuge tube. After collection of the whole blood, allow the
blood to clot by leaving it undisturbed at room temperature. This usually takes 15-30
minutes. Remove the clot by centrifuging at 2500 to 3000 RPM, 10 minutes in a refrigerated
centrifuge. The resulting supernatant is designated serum. Following centrifugation, it is
important to immediately transfer the liquid component (serum) into a clean microcentrifuge
tube using a pipette. The samples should be maintained at 2-8°C while handling. If the serum
is not analyzed immediately, the serum should be stored and transported at –20°C or lower. It
is important to avoid multiple freeze-thaw cycles because this is detrimental to many serum
components. Samples that are hemolyzed, icteric, or lipemic can invalidate certain tests.
Plasma Preparation –
Collect whole blood into commercially available anticoagulant-treated tubes e.g., EDTA-
treated. Cells are removed from plasma by centrifugation for 10 to 15 minutes at 3000 to
4000 RPM. The resulting supernatant is designated plasma. Following centrifugation, it is
important to immediately transfer the liquid component (plasma) into a clean
microcentrifuge tube using a pipette. The samples should be maintained at 2-8°C while
handling. If the plasma is not analyzed immediately, the plasma should be stored and
transported at –20°C or lower. It is important to avoid multiple freeze-thaw cycles. Samples
which are hemolyzed, icteric, or lipemic can invalidate certain tests.

Urine Specimen Collection
Supplies (sterile urine cup, clean catch wipes, plastic bag for transport if necessary)
can be obtained from the LAB or physician's office. Please follow these instructions:
• Clean genital area with the wipes.
FEMALES: Clean from front to back;
MALES: Clean urethral opening area.
• Allow first urine flow to go into the commode.
• Catch the "mid-stream" of the urine in the sterile container.
• Replace lid firmly on the container.
• Label specimen with name, date and time of collection.
• Place the specimen in the transport bag.
• Transport the specimen to the lab as soon as possible. If delay is necessary,
specimen may be kept in refrigerator up to 24 hours.

What is a 24-hour urine collection?
• A 24-hour urine collection is a simple lab test that measures what’s in
our urine. The test is used to check kidney function. A 24-hour urine
collection is done by collecting your urine in a special container over a
full 24-hour period. The container must be kept cool until the urine is
returned to the lab.
• Urine is made up of water and dissolved chemicals, such as sodium
and potassium. It also contains urea. This is made when protein breaks
down. And it contains creatinine, which is formed from muscle
breakdown. Normally, urine contains certain amounts of these waste
products. It may be a sign of a certain disease or condition if these
amounts are not within a normal range. Or if other substances are
present.

24-Hour Urine Collection
Preservative:
• Acid - Be very careful not to splash contents. Should an accident occur, rinse any
splashed areas with copious amounts of water. Submit specimen as soon as possible
and within 24 hours of collection, unless otherwise instructed.
• None - Keep specimen container in the refrigerator during the collection period and
until it can be delivered to the lab (as soon as possible but within 24 hours of
completion).
Procedure:
1.Empty the bladder completely on awakening in the morning. DISCARD THAT URINE.
2.Record the date and time.
3.All urine passed during the remainder of the day and night must be poured into the
container. Urine passed during bowel movements must also be collected but take care
not to contaminate the urine with the feces as this may adversely affect the test results.
4.The next AM, end the collection by Saving what urine you are able to pass at exactly the
same time as the previous AM.

Role of urine preservatives:
1. These preservatives are added to the following:
1. Reduce bacterial growth.
2. Decrease the decomposition of the chemicals.
3. Keep the substance in solute form.
4. Decreases the atmospheric oxidation of unstable compounds.
2. Refrigeration is the most useful for the collection and its utility increases with the
preservatives.
3. There are tablets commercially available are:
1. Potassium acid phosphate.
2. Sodium benzoate.
3. Benzoic acid.
4. Methenamine.
5. Sodium bicarbonate.
6. Red mercuric acid.

Examination of Urine
• Urine Analysis, Physical Examination
• These are the normal constituents and findings in normal urine.
• Routine urinalysis includes:
• Physical character.
• Chemical analysis.
• Microscopic examination.
• Precautions for urine collection:
1.Urine must be analyzed within one hour of collection if held at room temperature.
2.Keep urine at 2 to 8°C if delayed >8 hours.
3.If urine is kept for a longer time, then it will get a false positive test, like nitrite will be
positive.
4.Urea-producing organisms will degrade urea to ammonia and change the pH to alkaline.
5.Change in pH causes degeneration of cast and cell lysis.

Factors that will interfere with the urine analysis:
1. Certain foods will color the urine, like:
1. Carrots will change their urine color to dark yellow.
2. Beets may cause red color urine.
3. Rhubarb will change its color to red or brown.
2. Asparagus produces a typical urine odor.
3. If you keep urine on the table, bacteria will change the urine pH, making it alkaline because of urea-splitting
bacteria, and there is the production of ammonia.
4. Effect of the food on the urine pH:
1. Eating citrus fruits, dairy products, and vegetables makes urine alkaline.
2. A high protein diet and some foods (cranberries) make urine acidic.
3. A high protein, a fat diet without the intake of carbohydrates also causes ketonuria.
5. Females may have contamination from the vaginal secretions and show an increased number of WBCs.
6. Traumatic catheterization may cause hematuria and false RBCs in the urine.
7. Urine culture gives false-positive results when contaminated with stool, vaginal secretions, and contaminated
test tubes or hands and clothes.

Urine Physical examination:
A. Color
1. The color of the urine varies from colorless to black.
2. This variation in the color depends upon the following:
1. Metabolic functions.
2. Physical activity.
3. Ingested food.
4. Pathologic conditions.
3. The yellow color of the urine is due to pigment, which is called urochrome, derivatives of urobilin, the end
product of bilirubin degradation.
4. Color intensity depends upon the concentration of the urine. Concentrated urine is darker in color.
5. The pale yellow to light color is due to diluted urine.
6. Yellowish-brown to green color is due to bile pigments oxidation. While the blue/green color is due to
pseudomonas infection.
7. Blue urine color is due to the intake of medication of methocarbamol, methylene blue, and amitryptiline.
8. The reddish-brown color in the fresh sample is from hemoglobin and red blood cells.
9. Urine is positive for blood by chemical tests, the brownish-black color ion is standing in the urine due to
alkaptonuria because of the homogentisic acid excretion. In this case, the chemical blood tests will be negative.
13.Drugs and some foods like beets may change the urine color.
14.If urine shows large foam on shaking, it indicates an increased amount of protein in the urine.

B. Odor
1. This is not part of the urine analysis. It has no clinical significance.
2. Odor has little value in the diagnosis of various possibilities.
3. There is a typical ammonia smell.
1. When urine is sometimes kept on the table,
the odor of ammonia (NH 3) becomes more prominent.
2. The breakdown of the urea by the bacteria in the urine
is responsible for the ammonia smell.
4. Urinary tract infection gives a noxious, fecal smell, and it is unpleasant to smell.
5. Diabetic urine often smells fruity as a result of ketones.
6. Ingestion of onions, garlic, and asparagus can cause an unusual or pungent odor.
7.A bleach-like smell is seen in the contamination.
8. The mousy odor is seen in phenylketonuria.

C. Clarity
1.Normal urine is clear and is judged against the light source.
2.The cloudiness of the urine specimen depends on pH and dissolved solids components like
amorphous phosphates and carbonates.
Urine degree of clarity (cloudiness) Criteria
Clear No visible particulate material is seen.
Hazy 1.Can see visible particulate material
2.Can read the newspaper
Cloudy 1.Can see the newspaper
2.But the words are distorted or not
clear.
Turbid 1.Can not see the newspaper through
the urine tube

Is your Urine Cloudy?
Common Causes:
• Urinary Tract Infection
• Kidney Problems
• Prostate problems
• High ketone levels
• Pregnancy
• Dehydration
• STD

D. Turbidity:
1. It is generally due to gross bacteriuria.
2. In alkaline urine, turbidity is due to amorphous phosphates and carbonates.
3. In acidic urine, turbidity is due to amorphous urates.
4. Smoky urine is due to hematuria.
5. In women, the epithelial cells and mucus may result in hazy urine.
6. Urine, when kept in the fridge, may become turbid without any pathology.
• In refrigerated urine, amorphous phosphates, carbonates, and urates give rise to
thick turbidity.
7. Other turbidity causes are contamination with semen, feces, vaginal cream,
and contrast media.
8. Turbidity may be seen in bacterial infection and the presence of RBCs and
WBCs.

E. Specific gravity
Specific Gravity is a simple indicator of how concentrated the urine is. The
normal range of specific gravity in adults is 1 to 1.030. An increase in this
could be an indicator of dehydration, diarrhea, urinary tract infection,
heart failure or decrease blood flow to the kidney. A decrease in specific
gravity could indicate conditions such as renal failure or pyelonephritis or
excessive fluid intake.

F. pH
1. The lungs and the kidneys are major regulators of the acid-base balance of the body.
2. Urine pH must be performed on a fresh urine sample because the urine tends to change
the pH on standing.
3. The first-morning sample shows a pH of 5 to 6.
• After the meal pH may be alkaline.
• Urine pH is 4.6 to 7.0, and the average is 6.0
4. pH depends upon the diet.
5. pH <7.0 is primarily caused by the phosphates, which are excreted as salts conjugated to
Na+, K+, Ca+, and NH4+.
6. The acidity of urine is seen in the following:
• Systemic acidosis in diabetes mellitus.
• Renal tubular acidosis.
7. The alkaline urine (>7.0) is seen in:
• Urinary tract infection.
• Bacterial contamination of the urine.
• Medications like sodium citrate and sodium bicarbonate will induce pH.

G. Volume
1. The urine volume depends on the amount of water excreted by the kidneys.
2. The volume of the urine depends upon the following:
1. The fluid (water) intake.
2. Fluid (water) loss from nonrenal sources.
3. The amount of ADH secretion.
4. Excretion of dissolved solids such as glucose or salts.
3. The urine volume excreted indicates the balance between fluid ingestion
and water loss from the lungs, sweat, and intestine.
4. Normal urine volume:
1. 1200 to 1500 mL/24 hours.
2. The range of 600 to 2000 mL/24 hours may be considered normal.
3. The average urine volume is 1200 ml.
5. Night urine volume is usually less in amount.

1.Nocturnal polyuria:
1. There is increased urine at night. This may be seen in diabetes mellitus and diabetes insipidus.
2. This may be seen as diuretics or intake of tea, coffee, or alcohol. These will suppress the ADH.
2.Polyuria is seen in:
1. Diabetes mellitus.
2. Diabetes inspidus.
3. Chronic renal disease.
4. In the case of acromegaly.
5. In the case of myxedema.
3.Oliguria:
1. There is a decrease in the normal daily urine volume.
1. Anuria or oliguria, where urine volume is <200 mL/day.
2. This is seen in dehydration due to vomiting, diarrhea, perspiration, or severe burn.
3. Nephritis.
4. Urinary tract obstruction.
5. Acute renal failure.
6. Oliguria may lead to anuria.
7. Drugs that have diuretic effects are:
1. Thiazides.
2. Alcohol.
3. Caffeine.
8. The drugs which decrease the volume and are nephrotoxic are:
1. Analgesics like salicylates.
2. Antibiotics like neomycin, penicillin, and streptomycin.

Normal urine picture
Physical features Chemical features Microscopic findings
1. Color = Pale yellow or
amber
2. Appearance = Clear to
slightly hazy
3. pH = 4.5 to 8.0
4. Specific gravity = 1.015
to 1.025
1. Blood = Negative
2. Glucose = Negative
3. Ketones= Negative
4. Protein = Negative
5. Bilirubin = Negative
6. Urobilinogen = Negative
(±)
7. Leucocyte esterase =
Negative
8. Nitrite for bacteria =
Negative
1. RBCs = Rare or Negative
2. WBC = Rare or Negative
3. Epithelial cells = Few
4. Cast = Negative
(Occasional hyaline)
5. Crystal = Negative
(Depends upon the pH of
the urine)
6. Bacteria = Negative

The Chemical Examination of Urine
The chemical examination is usually performed in the laboratory using
urine test strips with rows of test pads called reagents. Each pad on the
urine test strip contains a different chemical that will change colour to
indicate the presence of specific substances in your urine. The colour
indicates whether a small or large amount of the substance has been
detected. The results are often read by the technician; however,
sometimes the reading is automated to prevent errors of interpretation
and timing since each pad color needs to be analyzed at the correct
time (ranging from a few seconds to minutes dependent on the
reaction it is detecting) to ensure accurate results.

What does the chemical examination test for?
The chemical examination can test for a range of indicators:
• Protein
• Blood (Haemoglobin)
• pH Level
• Leukocytes
• Nitrates
• Bilirubin
• Urobilinogen
• Ketones
• Glucose

A. Protein
• Protein in the urine is usually measured by testing for the presence of albumin, but there
is a range of alternative protein tests that can be used. High protein levels (proteinuria)
can be an early indicator of kidney disease. Detection of protein in the test pad may also
be an indicator of urinary tract infection/inflammation or injury such as damage to the
prostate, bladder or urethra. Proteinuria could also indicate conditions such as bladder or
kidney stones, multiple myeloma, or any condition that destroys red blood cells such as
haemolytic anaemia. It is often a temporary phenomenon that disappears after dealing
with an infection.
B. Blood (Haemoglobin)
• Haemoglobin is the protein found in red blood cells and carries oxygen. The urine
normally contains a small amount of haemoglobin which does not show up on the test,
but a higher amount (haemoglobinuria) will show up as a positive test result. It is
important that instructions are followed when collecting the sample, so that is not
contaminated through menstrual blood or blood from haemorrhoids. A slight increase
from the norm in haemoglobin can be significant in terms of potential causes such as a
urinary tract infection, kidney disease, trauma, strenuous exercise. Smoking or certain
medications can also show an increase.

C. pH Level
• The urine must be tested within a few hours of collection to avoid skewing the results for the pH
test, as urine will become more alkaline as time passes. The pH test tells us whether the urine is
acid or alkaline, this has a bearing where kidney or bladder stones (calculi) are involved. Certain
calculi are formed in overly-acid urine, some by overly alkaline urine. Diet largely affects the pH
balance of the urine, so this can be modified to reduce or eliminate stones or to hinder bacterial
growth. It is believed that a pH of over 7.5 will inhibit gram-negative bacteria from growing as fast.
• A vegetarian diet, a low carbohydrate diet or high-citrus fruit diet is associated with alkaline urine,
whereas high-protein or consumption of foods such as cranberries, meat or orange juice can
produce acidic urine.
D. Leukocytes
• Leukocytes is another name for white blood cells, of which there are several types. Much like with
haemoglobin, it is normal for urine to contain a few of these and the chemical test will show up
negative for a lower amount. However, a significant increase in leukocyte levels will give a positive
test which could be an indicator of inflammation of the kidneys/urinary tract or bladder/kidney
infection. Leukocytes and nitrites together suggest a UTI. White blood cells in your urine can
indicate that your kidneys/ureters or bladder or even other parts of your body have an infection
(viral or bacterial) and are inflamed. Inflammation does not automatically mean a bacterial
infection.

E. Nitrite
• The test for nitrite can be an indicator of the presence of a broad spectrum (but not
all) of bacteria that can cause a urinary tract infection since some harmful bacteria
convert nitrate to nitrite in the urine.
F. Bilirubin
• Bilirubin (a waste product created when old red blood cells are broken down) is not
normally present in healthy urine as it is usually removed as a component of bile. A
positive test, therefore, for bilirubin is an early indicator of hepatitis, liver disease or
jaundice.
G. Urobilinogen
• Urobilinogen is created in the intestine from bilirubin, and a proportion of it is
reabsorbed into the bloodstream. A positive test for urobilinogen can indicate
conditions such as hepatitis/cirrhosis of the liver. The urobilinogen result is also
compared with the bilirubin result. A negative test for bilirubin but positive for
urobilinogen can indicate haemolytic disease. A low or negative result for urobilinogen
in a patient with a positive bilirubin test can indicate a biliary/hepatic obstruction.

H. Ketones
• Healthy urine does not usually contain ketones. Presence of ketones in the
urine can indicate a lack of carbohydrates in the diet or that the carbs are
not being normally processed. Ketones indicate that fat is being
metabolised instead of carbohydrates for energy. This may be an
indication of frequent vomiting, a starvation diet, extreme exercising
regimes, or cold exposure. Raised ketones may also be an early indicator
of diabetes as it can point to insufficient insulin levels.
I. Glucose
• Any presence of glucose in the urine will give a positive test result.
Conditions indicated by glucose in the urine are uncontrolled diabetes,
kidney/hormonal disorders, liver disease. Glucose may also be an indicator
of pregnancy. A blood test is recommended to identify the specific cause
of a positive test result.

Microscopic Examination
• This test looks at a sample of the urine under a microscope. It looks at
cells from our urinary tract, blood cells, crystals, bacteria, parasites,
and cells from tumours, should they exist. This test is used to confirm
the findings of other tests or add information to a diagnosis, and
basically, it gives more specific information.
• The microscopic examination is usually performed if the physical or
chemical analysis shows abnormal findings. The urine sample is
centrifuged so that the concentrated substances can be isolated and
studied under the microscope where substances such as crystals and
cells can be counted.

A. Red blood cells
• A red blood cell number elevated from the norm can indicate injury, inflammation, disease/infection of the urinary
tract (e.g. bladder/kidneys/urethra).
B. White blood cells
• A white blood cell number elevated from the norm can indicate infection or inflammation of the urinary tract.
C. Epithelial Cells
• A raised number of epithelial cells from the norm can indicate infections, malignancies and inflammation of the
urinary tract. The types of cells found under the microscope can help to identify the area of the urinary tract where
there is a problem:
• transitional epithelial cells indicate a condition of the bladder
• squamous epithelial cells indicate a condition of the external urethra
D. Microorganisms
• Trichomonas: these are parasites found in the urine. If these are found, a further test for Trichomonas vaginalis is
often performed to investigate for vaginal infection.
• Yeast: indicates a vaginal yeast infection and requires a follow-up test on vaginal secretions (swab) to test for fungal
infection.
• Bacteria: these can enter the urethra from the exterior and travel up to the bladder causing a urinary tract
infection. If left untreated this can progress to a more serious kidney infection. Sometimes, the bacteria can
originate from inside the body, for instance in the case of septicaemia where the bacteria has infected the urinary
tract from the bloodstream. A follow-up urine culture test should be performed where harmful bacteria is found.

E. Casts
Casts are hot dog-shaped particles produced when the kidney cells secrete protein. Usually, these
are visibly clear (hyaline), but various kidney diseases alter their appearance, giving an indicator of
which disorder is present. For example, where red or white blood cell casts are found in the
microscopic examination, a kidney disorder is indicated.
F. Crystals
Normal urine contains a range of different crystals such as calcium oxalates, calcium carbonate,
crystalline uric acid, amorphous urate which are formed when waste chemicals bind together.
Crystals come in all kinds of shapes, colours and sizes. Whether a crystal form is dependent on how
acidic/alkaline your urine is (which can be affected by your diet), urine temperature and how much of
a particular substance is in your urine (since it is more likely to bind together). These ordinary
crystals cause no problem, but abnormal crystals in the urine can cause pain and damage to the
urinary tract such as cysteine, tyrosine and leucine. Kidney stones (calculi) form in the kidney and
can become lodged in either the kidney or ureters.

Examination of Body Fluids (Pleural, Peritoneal, Pericardial and Synovial)
Pleural Fluid Testing
• Pleural fluid is found in the thin space
between the lungs and the tissue covering
the lungs. Pleural fluid testing involves
removing a sample of this fluid so that it
can be analyzed using one or more
laboratory methods.
• Testing is used to diagnose the cause of an
abnormal buildup of pleural fluid, which is
called a pleural effusion. The fluid is
obtained by inserting a needle into the
pleural space, a procedure known as
thoracentesis.

Purpose of the test
• The purpose of testing pleural fluid is to determine the cause of a
pleural effusion. A pleural effusion occurs when excess fluid collects in
the area between the lungs and the tissue that covers the lungs,
known as the pleural space.
• While it is normal to have a small amount of fluid in the pleural space,
a pleural effusion involves an abnormal buildup of fluid that can
disrupt breathing and trigger other symptoms.
• The procedure to obtain a sample of pleural fluid is known as
thoracentesis, and it involves inserting a thin needle into the pleural
space to withdraw a small amount of fluid. When thoracentesis is
done for pleural fluid testing, it is called diagnostic thoracentesis. If
the procedure is done to reduce fluid buildup and relieve symptoms,
it is called therapeutic thoracentesis.

Procedure of taking a Pleural Fluid sample
• In general, the needle is inserted through the back while a patient is sitting up. Less
often, thoracentesis can be done while a person is lying on their back if they cannot
sit up or if the pleural effusion cannot be reached from the back.
• During a thoracentesis procedure, you are usually sitting upright and may be asked
to rest your arms on a table in front of you. Ultrasound is typically used to generate
an image of the location of the pleural effusion near your lungs.
• Once you are positioned, it is important that you do not move during the procedure.
At points during the procedure, you may be asked to exhale or hold your breath.
• While you are sitting up, the doctor identifies the specific area of your back where
they will insert the needle. That area is thoroughly cleaned with an antiseptic. Then,
a local anesthetic is applied to your skin in order to reduce pain.
• A needle is inserted through your skin and muscle and into the pleural space around
the lung. Fluid is then withdrawn through the needle. After enough fluid is collected,
the needle is withdrawn, and a bandage is placed over the puncture site.

What does the test measure?
Pleural fluid testing may involve multiple different analyses and measurements of the same test sample.
Common components of a pleural fluid test include:
• Visual examination: Looking at the pleural fluid with the naked eye can detect abnormalities in its
color, texture, and viscosity. Normally the pleural fluid is clear, yellowish (serous).
• Microscopic evaluation: A sample of the pleural fluid can be placed on a laboratory slide and
examined with a microscope. This can show the kinds of cells present, including germs like bacteria or
fungi that can cause infections.
• Cytology: Cytology is a detailed examination of the specific kinds of cells in the sample and is often
utilized to determine whether cancer cells are present in the pleural fluid.
• Bacterial or fungal culture: A culture test places a sample of the pleural fluid in a dish in the
laboratory where it is left for a period of days to see if any bacteria or fungi grow.
• Total protein : Total protein is the sum of all types of proteins in the pleural fluid and may be reported
as the concentration of protein in the sample.
• Lactate dehydrogenase (LDH): Lactate dehydrogenase is involved in normal cell activity but may be
produced at higher levels in response to inflammation or tissue damage.

Interpreting test results / Causes
• A transudative pleural effusion involves watery fluid that has leaked into the
pleural space. Transudative pleural effusions are most frequently caused by
heart failure or liver or kidney problems.(Clear fluid)
• An exudative pleural effusion involves protein-rich fluid entering the pleural
space. There are more diverse potential causes of exudative pleural effusions
including infections, inflammation, lung problems, tumors, and blockage of
blood or lymph vessels. (cloudy fluid cz cells, protein, cholesterol high)
• Infection: If bacteria or fungi are found in the pleural fluid, it is often a sign of
an infection. Pneumonia and tuberculosis are conditions caused by infections
that may be detected through pleural fluid analysis.
• Cancer: Testing may find cells that are cancerous, which can be the result of a
tumor somewhere in the body. Types of cancer most often found in pleural
fluid testing include breast cancer, lung cancer, lymphoma, stomach cancer,
and ovarian cancer.
• Empyema: Empyema is when pus enters the pleural space. Pus is a mixture of
immune cells, dead tissue, and germs. Empyema frequently occurs as a result
of an infection.

Peritoneal fluid Testing
• Peritoneal fluid is a liquid that acts as a lubricant in the abdominal
cavity. It is found in small quantities (generally 5-20 mL) between the
layers of the peritoneum that line the abdominal wall. Peritoneal fluid
acts to moisten the outside of the organs and to reduce the friction of
organ movement during digestion and movement.
• A variety of conditions and diseases can cause inflammation of the
peritoneum (peritonitis) and/or excessive accumulation of peritoneal
fluid (peritoneal effusion or ascites). Peritoneal fluid analysis is a group
of tests that evaluate this liquid to determine the cause of the increased
fluid.

The two main reasons that fluid may collect in the abdominal cavity are:
• Transudate—an imbalance between the pressure within blood vessels (which drives fluid out of
the blood vessel) and the amount of protein in blood (which keeps fluid in the blood vessel) can
result in accumulation of fluid (called a transudate). Transudates are most often caused by
congestive heart failure , nephrotic syndrome, or hepatic cirrhosis.
• Exudate—injury or inflammation of the peritoneum may cause abnormal collection of fluid
(called an exudate). Exudates are associated with infections, malignancies, pancreatitis,
ruptured gallbladder, or autoimmune disease. Laboratory tests may be performed to determine
one of the following conditions:
• Infectious diseases caused by viruses, bacteria, or fungi; infections may originate in the
peritoneum due to a rupture of the appendix, perforation of the intestines or the abdominal
wall, contamination during surgery, or an infection from other places in the body that has
spread to the peritoneum.
• Inflammatory conditions – peritonitis due to certain chemicals, irradiation, or rarely an
autoimmune disorder
• Malignancies – such as mesothelioma, tumor of the liver (hepatoma), lymphoma, or metastatic
cancer
• Pancreatitis – due to certain drugs, infections, blockages, and circulating lipids (fats)

Paracentesis Procedure:-
• Preparation: The patient is positioned lying down, and the abdomen is
cleaned. Local anesthesia is applied to numb the area.
• Needle Insertion: A thin needle or catheter is inserted into the
abdomen to access the fluid in the peritoneal cavity, often guided by
ultrasound.
• Fluid Removal: Fluid is drawn out through the needle into a syringe or
collection bag.
•Post-Procedure Care: The needle is removed, the site is bandaged, and
the patient is monitored briefly for any immediate complications. Fluid
samples may be sent for lab analysis.

What does the test result mean?
Transudate
• Most ascitic fluids are transudates and are caused by either congestive heart failure or hepatic cirrhosis. Typical fluid analysis results include:
• Physical characteristics—fluid generally appears clear or straw-colored
• Protein—less than 3 g/dL
• Albumin level—low (typically evaluated as the difference between serum albumin and peritoneal fluid albumin, termed serum-ascites albumin
gradient, or SAAG; values above 1.1 g/dL are considered evidence of a transudate.)
• Lactate dehydrogenase (LD) fluid/serum ratio—less than 0.6
• Glucose—equal to glucose level in the blood
• Cell count—few cells are present, usually lymphocytes
• Specific gravity—less than 1.015
Exudate
• Physical characteristics—fluid may appear cloudy
• Protein—greater than 3 g/dL
• Albumin level—higher than in transudates (typically with a SAAG less than 1.1 g/dL)
• Lactate dehydrogenase (LD) fluid/serum ratio—greater than 0.6
• Glucose—less than 60 mg/dL
• Cell count—increased
• Specific gravity—greater than 1.015

Physical characteristics – the normal appearance of a peritoneal fluid sample is usually straw-colored and clear.
Abnormal appearances may give clues to conditions or diseases present and may include:
• Yellow with liver disease, milky from obstruction of the lymphatic system, and greenish from bile
• Reddish peritoneal fluid may indicate the presence of blood, most often due to trauma.
• Cloudy peritoneal fluid may indicate the presence of microbes and/or white blood cells (WBCs), pointing to an
infection. It may also indicate lymph system blockage or trauma.
Chemical tests – tests that may be performed in addition to albumin may include:
• Glucose—typically about the same as blood glucose levels; may be lower with infection
• Amylase—increased with pancreatitis
• Tumor markers—to identify type of malignancy
Microscopic examination – may be performed if infection or cancer is suspected; normal peritoneal fluid has
small numbers of WBCs but no red blood cells (RBCs) or microbes. Results of an evaluation of the different kinds
of cells present may include:
• Total cell counts—WBCs and RBCs in the sample are enumerated. Increased WBCs may be seen with infections
and malignant conditions.
• WBC differential—determination of percentages of different types of WBCs; an increased number of
neutrophils may be seen with bacterial infections.
Cytology—a special centrifuge (cytocentrifuge) concentrates a small sample of cells that is then treated with a
special stain to be examined under a microscope for abnormal cells and for white blood cell differentiation. The
differential can help determine whether the cells are the result of an infection or the presence of a tumor.

Pericardial fluid test
• The pericardium is a two-layered, sac-like membrane that surrounds
the heart. Pericardial membranes produce pericardial fluid, a liquid
that sits between the pericardium’s membranes. The fluid acts as a
lubricant for the movement of the heart, reducing friction as the
heart pumps blood.
• A variety of conditions and diseases can cause inflammation of the
pericardium (pericarditis) and/or excessive accumulation of
pericardial fluid (pericardial effusion). Pericardial fluid analysis is a
group of tests that evaluate this liquid to help diagnose the cause of
the increased fluid.

Causes of fluid accumulation in the pericardial space are / Why
pericardial fluid is used to diagnose :-
• Transudate—an imbalance between the pressure within blood vessels (which
drives fluid out of the blood vessel) and the amount of protein in blood (which
keeps fluid in the blood vessel) can result in accumulation of fluid. Transudates are
most often caused by congestive heart failure or cirrhosis.
• Exudate—injury or inflammation of the pericardium may cause abnormal
collection of fluid (called an exudate). Exudates are associated with a variety of
conditions and diseases. If the fluid is an exudate, then several additional tests
may be ordered to help diagnose the specific condition, which may include:
• Infectious diseases – caused by viruses, bacteria, or fungi. Infections may originate in the
pericardium or spread there from other places in the body. For example, pericarditis may follow
a respiratory infection or a chest cold.
• Bleeding – bleeding disorders and/or trauma can lead to blood in the pericardial fluid.
• Inflammatory conditions – pericarditis may follow a heart attack, radiation treatment, or be part
of autoimmune disorders such as rheumatoid arthritis and lupus.
• Cancer – such as mesothelioma that has arisen in the pericardium or metastatic cancer that has
spread to it.

Signs and symptoms for pericardial effusion:
• Chest pain, sharp or sometimes dull, that may be relieved by bending
forward
• Coughing
• Difficulty breathing
• Fever
• Fatigue
• Changes in heart rhythm
• Enlarged heart size on chest X-ray
• Abnormal pericardial appearance on echocardiogram

What does the test result mean?
Transudate
• Transudates are most often caused by either congestive heart failure or cirrhosis. Typical fluid
analysis results include:
• Physical characteristics—fluid appears clear
• Protein or albumin level—low
• Cell count—few cells are present
Exudate
• Exudates can be caused by a variety of conditions and diseases. Initial test results may show:
• Physical characteristics—fluid may appear cloudy
• Protein or albumin level—high
• Cell count—increased

Additional test results and their associated causes may include:
Physical characteristics – the normal appearance of a sample of pericardial fluid is straw-colored and clear.
Abnormal results may give clues to the conditions or diseases present and may include:
• Milky appearance—may point to lymphatic system involvement.
• Reddish pericardial fluid may indicate the presence of blood.
• Cloudy, thick pericardial fluid may indicate the presence of microorganisms and/or white blood cells.
Chemical tests – in addition to protein or albumin, a glucose test may be performed. Glucose in pericardial fluid
samples is typically about the same as blood glucose levels. It may be lower with infection.
Microscopic examination – normal pericardial fluid has small numbers of white blood cells (WBCs) but no red
blood cells (RBCs) or microorganisms. Results of an evaluation of the different kinds of cells present may
include:
• Total cell counts—quantity of WBCs and RBCs in the sample. Increased WBCs may be seen with infections and
other causes of pericarditis.
• WBC differential—determination of percentages of different types of WBCs. An increased number of
neutrophils may be seen with bacterial infections.
Cytology—a cytocentrifuged sample is treated with a special stain and examined under a microscope for
abnormal cells. This may be done when a mesothelioma or metastatic cancer is suspected. The presence of
certain abnormal cells, such as tumor cells or immature blood cells, can indicate what type of cancer is involved.

How is the Pericardiocentesis procedure carried out?
• The patient should lie down on an exam table at a 60-degree angle. If the patient
experiences a severe drop in blood pressure or a slowed heartbeat during
surgery, the doctors may provide fluids or prescribe medications intravenously. A
numbing agent is applied to the skin below and around the breastbone. During
the procedure, the patient remains conscious.
• The pericardial sac is ruptured using a needle and then guided in with the help of
an echocardiogram. The patient may feel slight pressure during this procedure. It
allows the doctor to see a moving image of the heart, aiding in fluid drainage
monitoring. After needle insertion, the needle is replaced with a catheter. It
takes 20 to 60 minutes to complete the entire procedure.
• The catheter remains in the patient’s chest for a few hours to allow fluid to drain
into a container. Once the fluid is removed from the sac, the cardiologist
removes the catheter.

Synovial Fluid examination:
• Synovial fluid is the liquid that surrounds and lubricates joints. When
patients have swollen, painful joints, a synovial fluid analysis can give
valuable information about the underlying source of the problem.
• A number of tests can be conducted on synovial fluid after it is
extracted from the joint space during a procedure called
arthrocentesis. The appearance of synovial fluid, as well as its
chemical properties, microscopic composition, and other signs of
infectious disease may be analyzed.

Why Synovial Fluid examination is performed:
• Autoimmune disorders: Autoimmune disorders are conditions in which a
person’s immune system mistakenly attacks healthy tissue. In some
autoimmune disorders, including rheumatoid arthritis, lupus, and psoriatic
arthritis, the immune system damages healthy tissues of the joints.
• Septic arthritis: Septic arthritis is an infection caused by microbes such as
bacteria or fungi that lead to acute inflammation in one or more joints.
• Gout:or pseudogout: Gout and pseudogout are painful forms of arthritis,
caused by the accumulation of uric acid or calcium crystals in the joint.
• Bleeding in the joint: Also called hemarthrosis, an accumulation of blood
in the joint can be caused by an injury to the joint, cancer, hemophilia, and
other health conditions.

Sign and Symptoms :
• Joint swelling, often referred to as joint effusion
• Sudden pain and warmth in a joint
• Redness at a joint
• Difficulty bending a joint
• Joint discomfort accompanied by fever

Chemical Analysis of Synovial Fluid
Test Name What It Measures Why
Glucose Testing The amount of glucose Glucose levels are lower in
certain conditions, including
autoimmune disorders or
septic arthritis.
Lactate The amount of lactate High levels of lactate are
associated with infection.
Total Protein The amount of protein Elevated protein concentration
can indicate an inflammatory
or bleeding disorder.
Uric Acid Testing The amount of uric acid The presence of uric acid
indicates gout.

Microscopic Assessment of Synovial Fluid
Test Name What It Measures Why
White Blood Cell (WBC)
Count and Differential
Presence and type of WBCs WBC levels become
elevated in response to
infections, septic arthritis,
gout, and pseudogout.
Gram Stain Presence and type of
microbes
Positive Gram stains can
help diagnose septic
arthritis and infection.
Bacterial Culture Presence and type of
microbes
The presence of certain
microbes may indicate
infection.

Aspiration (Arthrocentesis)
• Site Disinfection: Clean the skin over the joint
with an antiseptic solution.
• Anesthesia (if needed): A local anesthetic
may be applied to numb the area, reducing
discomfort.
• Needle Insertion: Insert a sterile needle into
the joint space. The specific location and
depth depend on the joint size and
accessibility.
• Fluid Collection: Using a syringe, aspirate
synovial fluid from the joint cavity. Collect
enough fluid for multiple tests, if needed.

Cerebrospinal fluid (CSF)
• Cerebrospinal fluid (CSF) is a clear, colorless, watery fluid that flows in
and around our brain and spinal cord. Brain and spinal cord make up
our central nervous system. It controls and coordinates everything we
do, including our ability to move, breathe, see, think, and more.
• Cerebrospinal fluid acts like a cushion that helps protect our brain and
spinal cord from sudden impact or injury. The fluid also removes
waste products from the brain and helps central nervous system work
properly.
• A CSF analysis is a group of tests that use a sample of our
cerebrospinal fluid to help diagnose diseases of the brain and spinal
cord and other conditions that affect the central nervous system.

• Adult CSF volume is estimated to be 150 ml with a distribution of 125 ml within the
subarachnoid spaces and 25 ml within the ventricles.
• Ependymal cells, which create cerebral spinal fluid (CSF), line the ventricles of the
brain and central canal of the spinal cord.
Pathway of CSF:
• CSF is produced mainly by a structure called the choroid plexus in the lateral, third
and fourth ventricles. an adult produces between 400 to 600 ml per day.
• CSF flows from the lateral ventricle to the third ventricle through
the interventricular foramen (also called the foramen of Monro).
• The third ventricle and fourth ventricle are connected to each other by the cerebral
aqueduct (also called the Aqueduct of Sylvius).
• CSF then flows into the subarachnoid space through the foramina of Luschka (there
are two of these) and the foramen of Magendie (only one of these).

What is it used for?
A CSF analysis is used to measure different substances in our cerebrospinal fluid.
It may include tests to diagnose:
• Infectious diseases of the brain and spinal cord,
including meningitis and encephalitis. CSF tests for infections look at white
blood cells, bacteria, and other substances in the cerebrospinal fluid
• Autoimmune disorders, such as Guillain-Barré Syndrome and multiple
sclerosis (MS). CSF tests for these disorders look for high levels of certain
proteins in the cerebrospinal fluid.
• Bleeding in the brain.
• Brain tumors, including from cancers in other parts of the body that may spread
to the central nervous system.
• Alzheimer's disease, the most common form of dementia, which includes
memory loss, confusion, and changes in behavior.

Symptoms of a brain or spinal cord infection include:
• Fever
• Severe headache
• Seizures
• Stiff neck
• Nausea and vomiting
• Sensitivity to light
• Double vision
• Changes in behavior
• Confusion

Functions
• The CSF has many protective and metabolic functions.
• The CSF acts as a shock absorber, by providing a fluid buffer
and thus protecting the brain from injury.
• It provides neutral buoyancy that prevents the brain from
compressing the blood vessels and cranial nerves against the
internal surface of the bones of the skull.
• It removes by-products of metabolism and plays an
important role in the homeostasis and metabolism of the
central nervous system.

How the Test is Performed
Lumbar puncture (spinal tap) is the most common method to get a sample of CSF.
1. Preparation of the Patient
Positioning: The patient is asked to either sit up with their back curved forward or lie on their side in a fetal position,
with the spine arched.
2. Sterilization: The lumbar area is cleaned with antiseptic to prevent infection.
3. Identification of the Puncture Site
• The doctor locates the L3-L4 or L4-L5 interspace (lower lumbar region) by palpating the iliac crests as a landmark.
4. Local Anesthesia
• A local anesthetic is administered to numb the area.
5. Insertion of the Spinal Needle
• The spinal needle is carefully inserted through the skin, between the vertebrae, and into the subarachnoid space,
where CSF is located.
6. CSF Collection
• CSF begins to drip slowly from the needle. It is collected into sterile tubes for laboratory analysis. a sample of 1 to
10 milliliters (mL) of CSF is collected in 4 vials
7. Needle Removal and Bandaging
• The needle is gently withdrawn, and the puncture site is covered with a sterile bandage.
• In some cases, special x-rays are used to help guide the needle into position. This is called fluoroscopy.

Sputum Examination
• Sputum is a highly specialized watery, colorless and odorless product
of the respiratory tract (bronchia, trachea & lungs).
• Sputum is mainly composed of mucus and also certain cellular and
noncellular components of host origin.
• During expectoration, sputum gets contaminated with normal
bacterial flora and cells from pharynx and mouth.
• It is the most frequently received specimen from the respiratory tract.
• Both its collection and examination are advantageous as samples are
easily obtained and its cellular content is representative of the entire
respiratory tract.

Indications
Identification of causative agent or organism
associated with a particular suspected infection of the
lower respiratory tract like,
• Suspected tuberculosis.
• Pneumonia especially if severe or in an immuno-
compromised host.
• Pneumocystis carinii pneumonia in HIV-positive
patients.
• Suspected fungal infection.
• Chronic disease like bronchiectasis.
Cytological examination for the investigation of viral infections (viral inclusions in
cytomegalovirus and herpes simplex infections), fungal infection, asbestosis and malignant
cells.

Sputum Collection
• Sputum sample is ideally collected in the morning (since secretions accumulate
overnight).
• The mouth should be free of foreign matter and may be rinsed with filtered water
prior to collection.
• Sputum sample is collected in a sterile, clean, dry and wide-mouthed plastic
container with a securely fitting screw cap. The container should be of break resistant
plastic and leak-proof to prevent desiccation and aerosol formation, and should have
the capacity of about 30 ml.
• The patient is advised to take a deep breath 2-3 times filling his/her lungs, coughs
deeply, and spit into the plastic container.
• About 2-5 ml of sputum is collected.
• Sample consisting only saliva (watery appearance, clear, and foamy) is not acceptable
for laboratory investigations; in such case, another sample should be collected.

• In those patients who cannot produce
sputum spontaneously by deep
coughing, a specimen of sputum may
be induced. This is done by inhalation
of 15% NaCl spray or propylene glycol
for 20 minute which are aerosolized to
stimulate sputum production.
• The container, containing sputum
sample, is caped securely and labelled
properly.

Transport
• For microbiological examination of sputum, sample should be sent to
the laboratory immediately.
• If sputum is allowed to stand, rapid reproduction of contaminating
bacterial flora from the throat and oral cavity will occur leading to
incorrect results.
• Pathogenic organism especially Haemophilus influenza, do not survive
for a long time in the collected sample.
• Sputum sample for bacterial culture should not be refrigerated.

Sputum Concentration Method (Petroff’s Method)
• If the sputum is too viscous, The sputum is transferred to a sterile test
tube and equal amount of sterile 4% NaOH is added.
• The tube is incubated at 37degree C for 30 minutes with vigorous
shaking every 5 minutes.
• The mixture is centrifuged at 3000 rpm for 30 minutes and
supernatant poured off.
• The deposit is neutralized by N/10 HCL using a drop of phenol red as
indicator.

Physical Examination
Quantity:
• Large amount of purulent sputum is coughed out In bronchiectasis.
• Large amount of watery sputum with pink tinge suggests pulmonary oedema.
Appearance/color:
• White, viscid, mucoid: Asthma, tuberculosis.
• Frothy, pink, serous: Broncho-alveolar carcinoma.
• Clear or mucoid, grey, glassy, tenacious: Chronic bronchitis.
• Yellow due to pus/neutrophils: Acute lower respiratory tract (pulmonary) infections.
• Green: due to infection caused by pseudomonas bacteria.
• Rusty due to lysis of red cells: pneumonia (E.G. Pneumococcal) and pulmonary infarction.
• Bright red due to fresh blood: pulmonary tuberculosis, lung tumors, carcinoma lung , pulmonary
infarction.
• Black due to coal dust: coal workers, in heavy smokers.

Microscopic Examination
Staining of sputum: two to three smears are made on A clean dry glass
slides and are stained with:
• Leishman's stain or wright stain for differential count.
• Gram’s stain for microorganisms
• Ziehl-neilsen stain for acid fast bacilli.
• Special stains for fungi.
• Papanicolaou stain for study of malignant cells.
• Other stains (depends on the clinical/pathological features).

Gram’s stain for microorganisms
Pathogenic organisms found
in sputum are;
• Gram-positive:
Staphylococcus aureus,
Streptococcus pyogenes,
Streptococcus pneumoniae.
• Gram-negative: Klebsiella
pneumoniae, Haemophilus
influenzae, Yersinia pestis,
Pseudomonas aeruginosa

Ziehl-Neilsen stain for acid fast bacilli
• Ziehl-Neilsen-stained sputum smear is
considered as positive if 5000-10000
tubercle bacilli/ml are present in the
sputum.
• Possibilities of detection of tubercle bacilli
are increased if multiple sputum samples are
examined.
• Mycobacteria appear as bright red straight
or slightly curved rods against a blue
background.
• Minimum 100 fields are examined before
reporting the smear as negative.

Parasites found in sputum
• Larvae of Strongyloides and
roundworm.
• Entamoeba histolytica: Cysts or
the trophozoites may be found
when an amoebic liver abscess
ruptures into the lungs.
• Echinococcus granulosae: Scolices
and hooklets of the larval form
may be seen with the rupture of
the hydatid cyst of the lungs into
the bronchus.

Leishman stain or Wright stain for differential count
Normal sputum consists of a few neutrophils, few lymphocytes, carbon laden
macrophages, occasional eosinophils and red cells.

Papanicolaou stain for study of malignant cells
• Cytological examination of sputum is normally carried out for the
diagnosis of bronchogenic carcinoma.
• For cytological examination, early morning sputum sample is
preferred.
• A thin sputum smear is prepared on clean, sterile, grease-free glass
slide from a yellowish, greyish, opaque, or blood-tinged portion, or
from tissue fragments in sputum and stained with Papanicolaou
technique.

Sputum Culture
• Culture media is inoculated with a floccule of the purulent portion of sputum
for absolute identification of microorganism.
• Saliva is washed away from sputum with sterile normal saline in order to
reduce the amount of contaminating normal bacterial flora in the inoculum.
• Blood agar plate and chocolate agar are inoculated with the washed sputum.
The chocolate agar plate is incubated in an atmosphere of extra carbon
dioxide (CO2) and blood agar plate is incubated aerobically.
• After the incubation for 18 hours, inoculated agar plates are examined for
growth; if growth is not sufficient, incubation for further 24 hours is indicated.
• Lowenstein-Jensen medium is used for culturing Mycobacterium Tuberculosis.

Stool Examination
• A stool test is a diagnostic procedure that involves analyzing fecal
matter to check for signs of infection, inflammation, or disease.
• A stool test is a diagnostic tool that healthcare providers use to find
out why we are having stomach and any bowel movement issues. This
test can detect bacteria, viruses and other germs in our that can make
sick. Healthcare providers use stool tests to help diagnose
gastrointestinal diseases and colon cancer.

Who needs a stool test?
Doctors may recommend stool testing for people who experience
symptoms of gastrointestinal disease, such as:
• gas
• bloating
• abdominal pain or cramping
• constipation
• diarrhea
• changes in bowel habits
• blood or mucus in the stool

How to collect a stool sample:-
A doctor or nurse will explain how to collect a stool sample and provide a small plastic container.
The United Kingdom’s National Health Service (NHS) provides the following instructions for collecting a
stool sample:
Step 1: Fill out the container label with details such as name, date of birth, and the sample date.
Step 2: Place one of the following inside the toilet to catch the stool, ensuring that the stool does not touch
the inside of the toilet:
•a Plastic Container
•Some plastic wrap stretched over the rim of the toilet, beneath the seat.
Step 3: Use the plastic spatula that comes with the container to place a sample of the stool inside the
container, then screw the lid shut. In most cases, a person should aim to fill around one-third of the container.
Step 4: Tip the remaining stool into the toilet, then place the collection spatula in a plastic bag, tie it up, and
dispose of it in the bin.
Step 5: Wash the hands thoroughly with soap and warm water.
•After collecting the sample, a person should give it to the medical professional as soon as possible. Delaying
this step can cause bacteria within the sample to multiply and could affect the accuracy of the test.

Physical examination of stool:
Sample should be examined immediately after collection. Samples left
standing prolonged will deteriorate helminthes, Ovum, other parasites and
increase the numbers of monilia and bacteria which gives wrong results,
however the following aspects of stool should be examined:
(1) Quantity: the adult person excretions about 150-250 gm. /day of feces.
(2) Consistency and form: Normal stool is well formed. But in constipation
(Dehydration) the stool is solid (Hard) and the semi-solid (soft or loose)
seen when taking certain medications and laxatives. In abnormal cases
such as diarrhea and dysentery the stool appear liquid, or watery in
nature. In cholera the stools have a rice water appearance. In cases of
malabsorption of fats the stools are pale bulky and semi-solid.

(3) Colour:
1- Normal colours of stools are light to dark brown due to the Presence of bile pigments.
2- Dark black: In cases with bleeding into the intestinal tract the stools become dark tarry in
nature due to the formation of acid hematin. if the bleeding is in the small intestines. In case
of bleeding in large intestines or rectum stool color may be bright red due to fresh blood.
3- Red color: Resulted from eating certain colorful foods such as red beets.
(4) Blood:
1- If The blood is found on the outer surface of the feces this caused either by contamination
from menstrual cycle blood in women or bleeding hemorrhoids from the blood vessels.
2- Blood should be noted in stools if present as it is indicative of Ulceration or presence of
any other pathology like malignancy.
(5) Parasite: Stools may contain adult helminthes. Nematodes like Ascaris are easily visible as
their size is large. Hook worms and Proglottids of cetodes may also present. These may be
visible to the naked eye.

Microscopic examination of stool:
The laboratory diagnosis of most parasitic infections is by the demonstration of ova of the
parasite in the stools of the infected person. The stool is collected in a clean container. The
stool can be examined by the following techniques.
(a)Wet mounts examination. (b)Iodine examination.
(a) Saline wet mount examination: The stool is emulsified in normal saline and a large drop is
placed on a glass slide and is then covered with a cover slide. Then examined under a light
microscope, it is important to examine specimen under 10X objective lens at first to
observe large molecules, cells, ova and helminthes, then to the 40X objective to complete
the test. It is preferable to keep the condenser down and the intensity of the light low for
proper visualization of the ova and cysts. The thickness of the film should be such that one
is able to see the printed letters of the newspaper through it.
(b) Iodine examination: Iodine preparation leads to better visualization of morphological
details of ova and cysts as it stains the glycogen in them. However it has the disadvantage
that the live trophozoites of Entamoeba histolytica and other live parasites cannot be seen
as the iodine kills them. The examination instructions in normal saline must be followed
the same in iodine test.

Microscopic examination include the following:
(1) Pus cells: Observed in stool the same procedure as in urine.
(2) RBCs: Observed in stool the same procedure as in urine.
(3) Monilia: Observed in stool the same procedure as in urine.
(4) Protozoa:
(a) Entamoeba histolytica: To investigate the vegetative phase (trophozoite) and cyst, causing amoebic dysentery disease.
(b) Entamoeba coli: trophozoite + cyst Note: - most of children diarrhea less than 2 years cause by Entamoeba coli.
(c) Giardia lamblia, trophozoite + cyst, Cause watery diarrhea disease in children, especially.
(d) Balantidium coli, trophozoite + cyst, causing Balantidiasis in colon.
(5) Worms :
(a) Enterobius vermicularis (pinworm): investigating the eggs that are of convex and flat surface and a pointed end.
(b) Ascaris lumbricoides: investigating for eggs which characterized by the content of granular yellow to Brown irregular
albumin membrane.
(c) Hookworm (Ancylostoma duodenale): investigating the eggs where the egg yolk is divided and surrounded by a thin
membrane.
(d) Tapeworms, (Taenia solium): investigating the worm pieces called (gravid segments or Proglottids) that comes out with
the feces.
(e) Schistosoma mansoni: Investigating the eggs distinct by lateral spin.

Chemical examination of stool
(a) pH: The pH of stools is acidic in amoebic dysentery and is alkaline in
bacillary dysentery.
(b) Occult blood: Presence of blood in feces which is not apparent on gross
inspection and which can be detected only by chemical tests is called as
occult blood. Causes of occult blood present in a number of diseases
including malignancy of the gastrointestinal tract. The reagents used are:
1- Benzidine reagent: - Development of blue colour is indicative of
presence of occult blood in the stool specimen.
2- Orthotolidine: Development of green colour Benzidine test is also highly
sensitive and false-positive reactions are common. Since bleeding from the
lesion may be intermittent, repeated testing may be required.

Stool culture Test
• The stool culture is a test that detects and identifies bacteria that cause infections of the lower digestive tract. The test distinguishes between
the types of bacteria that cause disease (pathogenic) and the types that are normally found in the digestive tract (normal flora). The test
helps to determine if pathogenic bacteria are the cause of gastrointestinal symptoms (gastroenteritis).
• A stool culture is used to detect the presence of disease-causing bacteria (pathogenic) and help diagnose an infection of the digestive tract. It
is often used along with other tests such as an Ova, Cyst and Parasite test that detects parasites in the stool and/or Clostridium difficile toxin
tests, to help determine the cause of prolonged diarrhoea. In establishing the cause, the test can help guide treatment, determining whether
antibiotics or other medication may be necessary to resolve symptoms.
• If a stool culture is positive for pathogenic bacteria, then they are the most likely cause of the diarrhoea and other symptoms. Results are
frequently reported with the name of the pathogenic bacteria that was detected. Laboratories typically evaluate stool cultures for the most
common intestinal pathogens:
• Campylobacter species
• Salmonella species
• Shigella species
Depending on the affected person's medical and/or travel history, tests for other pathogens or potential pathogens may be included or be
separately available. Some of these include:
• Aeromonas
• Plesiomonas
• Shiga-toxin producing Escherichia coli
• Yersinia enterocolitica
• Vibrio species

Module 3 - Clinical Pathology Sujoy Tontubay.pptx

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