Objective : To study and draw the various parts of microscope.

Microscope is an instrument which produces greatly enlarged images of small objects.

There are two types of microscopes:

1.Simple microscope
2.Compound microscope

Compound microscope has a series of lenses. One type of lens remains near the
object and is called objective lens and another type of lens is called eyepiece lens as it is
closer to the observers eye .They have varying magnifications .Compound microscope
can be either
1. Monocular-having single eyepiece
2. Binocular having two eyepieces

Light microscope has the following parts-

1. Support system
2. Magnification system
3. Adjustment system
4. Illumination system

Support system :
1. Stand- Gives stability to the microscope.
2. Body- Consists of a limb attached to a joint for positioning the microscope.
Body carries three parts which are-
a. Body tubes
b. Stage- is like a platform on which slide is kept. It has an aperture in centre through
which
light reaches object.
c. Revolving nose piece

Magnification system:
Consists of group of lenses-
1.Eyepiece- lenses at upper end of body tubes. Their magnification can be 5X, 10X or
15X.
2.Objectives- lenses present at bottom of the body tube. These have magnification power
of 10X, 45X & 100X (oil immersion).

Illumination system:
1.Light source
2.Mirror- reflect rays from light source onto object.
3.Condenser- Brings rays of light to common focus on object.
4.Diaphragm- Used to reduce or increase the angle of light ,thereby regulating the
amount of light passing through condenser.

1
Adjustment system:
1. Coarse adjustment screw
2. Fine adjustment screw
3. Condenser adjustment screw
4. Iris diaphragm lever
5. Mechanical stage controls.
Magnification power of microscope :It is the degree to which the image is enlarged .It
depends on :
1.Length of optical tube .
2.Magnifying power of objective lens.
3.Magnifying power of eyepiece lens.
Most of the standard microscopes have a fixed tube length of 160 mm ,hence the
magnification power of the microscope is
Magnifying power of objective x Magnifying power of eyepiece
.
How to use light microscope:
1.Keep microscope in a stable position.
2.Obtain appropriate illumination by adjusting mirror or intensity of light.
3. In case of unstained specimen, condenser should be at lowest position & iris
diaphragm closed or partially closed.
4. When using oil immersion lens, 100x objective should dip in oil (cedar wood oil)
5. Always clean the objective lens with tissue paper or soft cloth after using oil.

Problems while using microscope:

1. Light too strong Lower condenser
2. Light too faint- Adjust mirror.
-Use condenser of higher aperture.
3. Failure to focus under oil immersion .
-Quick movement of coarse adjustment .
-Viewing wrong side of slide.
-Too thick a coverslip.

Special types of microscopes:

1. Dark ground illumination (DGI)- It is used for examination of unstained living
microbes .e.g.-Treponema pallidum- causative organism of syphilis.
2. Phase contrast microscope- It enables observation of entirely transparent objects
without staining them.
3. Polarising microscope Is used for demonstration of birefringence e.g.apple green
birefringence of amyloid , crystals etc.
4. Fluorescent microscope- Is used for demonstration of microbes after staining them
with fluorescent dyes. e.g.- Mycobacterium tuberculosis by Auramine Rhodamine
,Fluroscent isothiocyanate etc.
5. Electron microscope-Is used for study of ultrastructural details of tissue and cells
.Tissue is fixed in 4% glutaraldehyde at 4 degree celsius and ultrathin sections are cut .
References:
[Basics of health science (WHO)]

2
Objective : To Study the various histopathological techniques.

Introduction :Histopathology is examination of tissue for presence or absence of

changes in their structure due to disease processes by examining thin sections of tissues
which are coloured differently by different stains.
Biopsy is the technique to excise small part of living tissue for the purpose of diagnosis
by histopathological examination .

Types of biopsy:
1.Incisional A small piece of diseased tissue is removed with the help of sharp knife .
2.Punch It is done with the help of punch biopsy forceps . e.g. cervical punch biopsy.
3.Currettage- It is the process of removal of tissues from cavities e.g. uterine cavity.
4.Drilling It is done for bony tissue .
5.Needle biopsy- An OPD procedure in which a linear tissue is obtained with the help of
fine cutting needle like liver biopsy , kidney biopsy .
6.Excisional biopsy It is done when whole diseased tissue is removed in toto like
mastectomy , hemimandibulectomy.

Steps of tissue processing :

1. Fixation.
2. Grossing
3. Dehydration.
4. Clearing.
5. Impregnation.
6. Embedding and blocking.
7. Section cutting.
8. Routine staining and mounting.

1.Fixation: It is the method of preserving cells and tissues as near to life like conditions
with the help of fixatives which act by denaturation or
precipitation of cell proteins or by making soluble components of cell insoluble.
Fixatives produce the following effects-
1.Prevents putrefaction and autolysis.
2.Hardens the tissue which helps in section cutting.
3.Make cell insensitive to hypertonic/ hypotonic solution.
4.Acts as a mordant.(facilitate the staining reaction)
5.Induce optical contrast for good morphologic examination.
Properties of ideal fixative-cheap,refractive,non-toxic & should facilitate the staining.

Commonly used fixatives are :

1. Formalin ( 10% )
2. Glutaraldehyde (2% )- used in electron microscopy .
3. Picric acid (bouins fluid)- used in testicular and renal needle biopsies and for

3
 demonstration of glycogen .
Disadvantage-It hardens the tissue & lysis of RBC
4. Carnoys fixative ( alcohol ) for cytologic smears and endometrial currettings _ gives
excellent nuclear fixation.
5. Osmium tetraoxide for CNS tissues and for electron microscopy which is the best
fixative for lipids .

2.Grossing : The gross description as recorded on the requisition form is a permanent

record of what was received from the operating room. The general format of the gross
description should include-
1. Name of the organ
2. Size
3.Appearance
4. Colour
5.Consistency
6.Any identifiable pathology
Blocks from the representative areas must be taken of proper size and thickness . Bone
and other calcific tissues are sent for decalcification to soften the tissue so that it is easy
to cut.
Decalcification-
It is the process of removal of calcium salts to soften the tissues like bone which
is carried out by treatment with reagents like 5-10% solution of nitric acid and
hydrochloric acid ,formic acid (10%) which react with calcium to form soluble calcium
salts or chelating agents like EDTA which take up calcium ions .

3..Dehydration :
It is the process in which water from cells and tissues is removed so that this
space is taken up by wax .Dehydration is carried out by passing the tissue through a
series of ascending grades of alcohol 70% ,80% ,95% and absolute alcohol to prevent the
shrinkage of tissue which occurs with rapid dehydration .If ethyl alcohol is not
available ,then methyl alcohol ,isopropyl alcohol or acetone can be used .

4..Clearing:
This is the process in which alcohol from tissues and cells is removed and is
replaced by a fluid in which wax is soluble and it also makes the tissue transparent .e.g.
xylene ,chloroform.

5.Impregnation:
This is the process in which empty spaces in the tissue and cells after removal of
water are taken up by paraffin wax .This hardens the tissue which help in section
cutting .It is done in molten paraffin wax(58-60 degree Celsius) .Volume of wax is 20-50
times more than the tissue.

Routine Manual processing in our lab:

Formalin -10 %-2 hrs minimum
Alcohol- 70% - 1hr

4
Alcohol- 80%- 1hr
Alcohol- 90%- 1 hr
Absolute alcohol I- 1.5 hrs
Absolute alcohol II-1.5 hrs
Absolute alcohol III-1.5 hrs
Xylene I- 0.5 hr
Xylene II-1 hr
XyleneIII-2 hr
Wax 1st - 2hrs
Wax 2nd 4hrs

Now a days all the processes of fixation , dehydration ,clearing and impregnation
are carried out in an automated tissue processor .

Advantages of automated tissue processor:

1. When workload is more
2. No supervision
3. Processing is uniform & reliable.

Disadvantage:
-Requires continuous power supply

6..Embedding And Blocking :

Embedding of tissue is done in molten wax , blocks of which are prepared using
L-mould ( Leuckharts mould ).The moulds are placed over a smooth surface / glass
tile .Molten wax is poured in the space between the L moulds. The processed tissue
pieces are put into the wax with the number tag and examining surface facing downwards
.
Wax is allowed to solidify .After solidification ,L-moulds are removed and each block
contains a tissue piece carrying a label .
Nowadays automatic embedding station is used for block making.

7.Section Cutting:
This is also known as microtomy .Microtome is an equipment for cutting thin
uniform section of tissue . Types of microtomes are-
I. Rotary
II. Sliding
III.Freezing
IV.Rocking
V.Base-sledge

Rotary microtome:It is the most commonly used microtome.In this microtome knife is
fixed while the tissue block movable.Knife in this faces upwards and is wedge shaped .
For routine work the thickness of sections recommended is 4-5 um.

Sharpening of knife:Two procedures -1.Honing

5
 2. Stropping.

7. Staining And Mounting:

Routine staining is done by Haematoxylin & Eosin stain .Haematoxylin is
obtained from the bark of tree Haematoxylan campechianum.
a. Deparaffinise section by xylene
b. Bring section to water by passing through descending grades of alcohol( as
Hematoxylin is water based dye)
c. Hematoxylin for 8-10 minutes
d. Rinse in water
e. Differentiation (that is,selective removal of excess dye from the section ) is done
by putting the slide in a solution of 1 % acid alcohol for 10 seconds.
f. Rinse in water
g. Blueing ( i.e. Bringing of required blue colour to the section , done by putting the
section in scotts tap water containing NaHCO3 and MgSO4 or saturated
solution of LiCO3 for 2-10 minutes).
h. Counter stain with 1% aqueous solution of eosin for 1-3 minutes.
i. Rinse in tap water.
j. Dehydrate the sections by passing in a series of ascending grades of alcohol.
k. Clearing in xylene.
l. Mount in DPX ( Distrene-80 dibutyl phthalate xylene )or Canada Balsam.
Results:
Haematoxylin ( Basic dye ) stains cell nuclei blue while eosin ( acidic dye) stains the
cell cytoplasm and the connective tissue fibres pink.

Frozen Section:
Frozen section cutting is a quick diagnostic procedure for tissues before proceeding
to a major radical surgery. This is also used for demonstration of some special substances
in the cells and tissues e.g.- fat , enzymes etc. This procedure can be carried out in O.T.
complex.

Merits:
1. It is a quick diagnostic procedure taking about 10 mins.
2. There is minimal shrinkage of tissue.
3. Lipids and enzymes can be demonstrated.

Demerits:
1. It is difficult to cut serial sections .
2. It is not possible to maintain tissue blocks for future use.
3. Sections cut are thicker in nature.
4. Structural details tend to be distorted due to lack of embedding media.

There are 2 methods for obtaining Frozen Section-

1.Freezing microtome using CO2 gas.

6
2.Refrigerated microtome.(cryostat).
Best results are obtained from fresh unfixed tissue and freezing the tissue as rapidly as
possible.
Special Stains:
1. Stains used for demonstration of fat are Oil red O,Sudan Black.
2. Stains used for demonstration of glycogen and mucopolysaccrides PAS.Other
PAS positive substances are colloid,neutral mucin and hyaline casts.
3. For demonstration of amyloid Congo red stain with polarized microscopy
4. Stain used for demonstration of iron -Prussian Blue ( Pearls Reaction.)

Cytology
It is the study of cells. It is of 2 types-
1. Exfoliative:
-Naturally shed eg. cells in sputum & urine
-Direct scrape eg. pap smear, bronchial brush
-Aspiration of fluid- eg. pleural fluid,ascitic fluid, synovial fluid ,CSF.

2 Fine needle aspiration cytology:

a. Direct
b. Guided
It is obtained from organs or any swelling (tumorous or inflammatory ) which are
easily accessible and can be aspirated by FNAC. All the specimens should be
accompanied by properly filled requisition forms, duly numbered and entered in the
register.

Examination of fluids:
1. Physical examination:
a. Quantity
b. Colour
c. Turbidity
d. Coagulum

2. Microscopic examination:
1.Total cell count of fluid is done by Neubauers chamber.
2.Counting is done by RBC & WBC method depending upon the number of cells.
3.Differential count is done by centrifugating the fluid.

Common techniques used for processing fluids are:

1. Preparation of sediment- smears from centrifuged samples-
a. Fluid is centrifuged for 10 min at 1500 rpm
b. Discard supernatant and put the button on slide with the help of glass pipette.
c. Fix in absolute alcohol.
2. Membrane filter preparation
Centrifuge and membrane filter preparation are useful for small routine fluids of low cell
count .

7
Staining procedure normally employed are :
1. Papanicolaou stain
2. H&E
3. Romanowasky stain like Leishman, May Grunwald Geimsa & Wrights stain

Museum technique:
Purpose :
a. Permanent exhibition of pathological condition for undergraduates and
postgraduates.
b. Collection of rare and interesting specimens.
c. Collection of specimens used in medical examinations,viva,lectures.
d. Permanent source of histopathological and photographical material for teaching
,research and publication.

Mounting of museum specimen

Method:
1. Fix the specimen according to lesion and make the cut surface smooth and even.
2. Mount the specimen on a plate and keep in Kaiserling solution I for 20-30 days.
3. Change to kaiyserling solution II and keep in it for -4 hours depending on size of
specimen.
4. Wash it in tap water.
5. Change to Kaiserling solution III and keep in it for 1 month.
6. After 1 month if the solution turns turbid change the solution.
7. Seal the lid with permanent sticking material.
8. Label the specimen according to system wise and lesion wise.

Kaiserling solution I (Also called fixing fluid)-

Formalin-400 ml
Potassium Nitrate-30gm
Potassium acetate-60 gm
Tap water-2L
.Discard the solution after single use.

Kaiserling solution II
It is prepared from 80 % propyl alcohol.
It can be reused once or twice till it remains clear.
It is used for restoring colour.

Kaiserling solution III

Glycerine 300ml
Sodium acetate 100gm
Formalin-5ml
To this mix tap water to make it 1 litre

8
References:
Cullings cellular pathology technique 4th edition.

Objective :To collect the sample of blood.

Sample collection: For investigation of blood,it is withdrawn predominantly by two

methods.
1.Capillary blood (Peripheral blood)
2. Venous blood

Capillary blood:

It is liable to give erroneous results and should be used only when it is not possible to
obtain venous blood.

Capillary blood can be obtained by

9
Adults:
1.Pricking of finger.
2.Lobe of ear
Infants:
1.Ball of thumb
2.Great toe
3.Heel(lateral or medial parts of the plantar surface. Central plantar area and posterior
curvature should not be punctured in small infants to avoid injury to underlying tarsal
bones)

Procedure:
1. Clean the area with spirit swab and let it dry .
2. Puncture should be about 3mm deep,wipe off first few drops of blood ,never press out
blood i.e. never squeeze finger .
3. Having obtained the requisite amount of blood,let the patient apply slight pressure
over the area with sterile swab.

Precautions:
1.Oedematous or congested part should not be used.
2.If the area to be puncture is cyanotic and cold ,warm it by massaging or else erroneous
result may be obtained.

Collection of capillary blood for quantitative studies:

Use a micropipette to draw up the correct amount of blood(usually 20l)

Uses:
1. Hb estimation
2. RBC count
3. Leucocyte count

Site:
This is best withdrawn from an antecubital vein by means of dry glass syringe or
disposable plastic syringe. The needles should not be too fine or too long. Those of 19 or
20 SWG are suitable and short needles with shaft 15mm long are particularly valuable for
children.
Nearest equivalent American gauges and diameters:
16SWG=14(1.625mm)
19SWG=18(1.016mm)
20SWG=19(0.914mm)
23SWG=22(0.610mm)

Venous blood:

Procedure:

10
Successful venepuncture can be done by-
1. Keeping the subjects arm warm
2. Applying tourniquet/sphygmomanometer cuff kept approximately at diastolic
pressure
3. Inspecting the veins in antecubital area
4. Ask the patient to open and close his fist several times.
5. Take aseptic precautions and puncture the vein.
6. Having withdrawn the blood, loosen the tourniquet ,apply a sterile guaze piece with
gentle pressure to stop oozing of blood.
7. Transfer the blood from syringe into container gently (not through the needle )

Differences between capillary & venous blood :

1. The PCV, Red cell count & haemoglobin content of capillary blood are slightly
greater than in venous blood.
2. TLC & neutrophil counts are high by about 8% & monocyte counts by about 12%
are higher in capillary blood
3. Platelet count appears to be higher in venous than in capillary blood.

Note-For CBC (complete blood count ),1.2 ml of blood in EDTA vial.For ESR 1.6 ml of
blood in citrate vial.(by Westergrens method )

Objective :To study the various anticoagulants used in haematology.

1. EDTA(Ethylene Diamine tetraacetic acid)

Sodium and potassium salts of EDTA in preferred for hematological purposes.

Mechanism: It acts by chelating calcium ions and preserves the cellular elements. To
achieve this, recommended concentration of 1.2 mg(approximately 4mol)of the
anhydrous salt per ml of blood is required.
Excess of EDTA affects both red cells and leucocytes,causing shrinkage and degenerative
changes

Uses:
1.CBC
2.PCV(packed cell volume).

11
2.Citrate
3.8%trisodium citrate

Mechanism: It acts by chelating calcium ions is used for ESR and coagulation studies.
PT and APTT.

Uses:
1.ESR (ratio is 1volume of citrate:4 volumes of venous blood)
2.Coagulation studies(1 volume of citrate:9 volumes of venous blood)
3.PT and APTT

3.Heparin (powder or liquid ):

1.Used at concentration or 10-20IU/ml of blood.
2.Effective anticoagulant and does not alter the size of the red cells
3.Heparinized blood should not be used for making blood films as it gives a faint blue
colouration to the background when the films are stained by Romanowsky dyes.
4.Should not be used for leucocyte counts as it tends to cause the leucocytes to clump.

Mechanism: acts by inhibiting thrombin and other stages of clotting factor activation in
presence of cofactor antithrombin III

Uses:
It is anticoagulant of choice for:
1. Osmotic fragility test
2. ABG ( Arterial Blood gas analysis)
3. Karyotyping.

4. Double oxalate:
It has potassium oxalate and ammonium oxalate used together in 2:3 ratio respectively.
Take 1% solution of potassium oxalate 0.4ml and 1% solution of ammonium oxalate
0.6ml in a test tube. Evaporate to dryness in the incubator.This amount is sufficient to
prevent coagulation of 5ml of blood.

Mechanism: acts by chelating calcium.

Uses:
1. For blood chemistry
2. PCV
3. 5 CPDA (citrate phosphate dextrose Adenosine)
4. Uses in blood banking. 14 ml of CPDA is used for every 100ml of blood that is 49 ml
of anticoagulant is used for 350 ml of blood (1 unit)

12
6. Plane vial:
Used to obtain serum sample for blood chemistry.

7.Fluoride:

Mechanism: prevents glycolysis by blocking phosphorylase enzyme in the RBCs

Uses : Anticoagulant of choice for determination of blood sugars.

8.Vacutainer / Evacuated tube system:

Sterile, disposable blood collection system meant for single use. It is simple to use,
quicker, cleaner and safer as it prevents infection.

References:
1) Practical Pathology. Dacie and Lewis. Eighth edition
2) A Handbook of Medical Laboratory Technolgy. V.H.Talib. second edition

13
Objective :To estimate the hemoglobin of the given blood sample.

Introduction : Hb is a conjugated protein containing 4 iron containing heme molecules

attached to the the globin molecule which is composed of 2 and 2 chains in normal
adult hemoglobin (Hb A2). It is the main component of R.B.Cs.The primary function of
hemoglobin is to carry oxygen from lungs to tissue cells and CO2 back from the cells to
lungs .

Collection of sample:
EDTA- Anticoagulated venous blood is commonly used .Other anticoagulants that can be
used are heparin and double oxalate. Capillary blood can also be used directly.

Methods of estimation :
1. Calorimetric methods
I.Visual :Sahlis Acid Hematin method
II.Photocalorimetric :
a.Alkali Hematin method
b.Oxyhemoglobin method
c.Cyanmethemoglobin method
2. Gasometric methods
3. Specific gravity methods
4. Chemical method
5. Cell coulter method

Sahlis Acid Hematin Method :

Principle : Hb is converted to acid hematin by the action of HCl, the colour of which is
compared against the glass rods of the standard comparator box.

Reagent and apparatus required :

N HCl, Sahlis Hemoglobinometer, Hb pipette, Distilled water .

Procedure :
1. Fill the hemoglobinometer tube with 0.1 N HCl. till the 2gm% mark.
2. Draw 20 ul blood into hemoglobin pipette.
3. Wipe the outside of pipette with gauze piece.
4. Blow the blood into acid solution in tube.
5. Allow the mixture to stand at room temperature for 10 mins.
6. Add 0.1 N HCl or distilled water drop by drop and stir with a glass rod after addition
of each drop and compare the color of tube contents with glass rods of the comparator
box. Stop when colour matches.
7. Note the level of upper meniscus of the fluid column and read the result directly in

14
 gm%.
Advantages:
1. It is an easy and inexpensive technique.
2. It does not require a calorimeter and hence can be done at bed side.

Disadvantages :
1. Since it is a subjective colour matching technique , there may be visual error in
matching .
2. After 10 mins, the color of acid hematin starts fading and a lower Hb value is
obtained if the reading is not taken in time.
3. The colour of the comparator fades over the years.
4. Acid does not convert all Hb into acid hematin. Therefore, the Hb value is slightly
lower than the actual value of Hb.( Carboxy Hb and Oxy Hb are converted and Sulf
and Meth Hb are not converted ).
5. Hb values below 2 gm% cannot be measured.

Cyanmethhemoglobin Method (To be demonstrated) :

Introduction : This is the most accurate method of hemoglobin estimation as it detects

all forms of hemoglobin except sulfhemoglobin. This is the method recommended by
WHO for hemoglobin estimation.

Reagent required:
The diluent used is Drabkins solution.
Contents of drabkins solution:
1.Potassium Ferricyanide -200 mg
2.Potassium cyanide -50 mg
3.Potassium dihydrogen phosphate 140 mg
4.Non-ionic detergent 1 ml
5.Water upto 1 litre

Principle: Hemoglobin is converted to cyanmethemoglobin by Drabkins solution and

the color of this solution is measured by a calorimeter and compared against a standard.

Advantages:
1. The compound formed is stable, hence large batches of samples can be studied.
2. Personal variation due to visual error is avoided as the reading is taken by
calorimetry.
3. It detects all forms of hemoglobin except sulfhemoglobin .

Disadvantages:
1. The turbidity of blood due to any reasons (eg high TLC) gives a falsely high Hb
value.
2. Hyperbilirubinemia effects the value.
3. KCN can be hazardous.(Lauryl sulphate, which is safe can be used in its place).

15
 Other Methods :

1.Specific gravity method for estimation of Hb.

Principle: A series of solutions of CuSO4 of known specific gravity are used to
compare the specific gravity with drop of blood from thr sample to be estimated.
CuSo4(at 1.053 specific gravity):- Hb > or = 12.5gm/dl
2. Alkali hematin method .
3. Gasometric method
4. Sheard Sanford oxyHb method .
5. Chemical method .
6. Direct reading hemoglobinometer
7. Spectrophotometry.

Normal values:
Men - 152gm% 1
Women - 13.51.5 gm % 1
At birth - 18.54 gm % 1
Children - (upto 6 yrs) -12.51.5 gm% 1

Clinical Significance:
1. Decrease in Hb concentration below normal values is known as anaemia.
2. Increased Hb value- ( more than 17 gm % ) is observed in Polycythemia which can be
a. Primary (neoplastic): Polycythemia rubra vera.
b. Secondary erythrocytosis
(i) in hypoxic patients: high altitude, pulmonary disease, congenital heart disease,
smoking, chronic pulmonary disease.
(ii) in patients with increased erythropoietin secretion: kidney tumors, cigarette
smoking , hypoxia including and congenital heart disease.
c. Spurious polycythemia: due to dehydration, decrease in plasma volume.

References:

1. Dacie and Lewis Practical Hematology; 9th edition; pg 12,13

16
Objective :To measure Erythrocyte sedimentation rate (ESR) of given blood sample.

Definition :
ESR is defined as rate at which erythrocytes sediment at their own weight in the given
interval of time for which anticoagulated blood is kept for one hour in ESR tube.
Sedimentation has 4 stages:
1. Rouleaux formation This occurs in first 15 minute & minimum sedimentation
occurs.
2. Foramtion of fine threads This occurs in next 15 minutes because of globulin and
fibrinogen.
3. Rapid fall Protein network along with red cell mass falls in next 15 minutes.
4. Packing phase Packing of RBCs occurs during the last 15 minutes.

Methods of Estimation :
Two methods commonly employed are :
1.Westergrens method
2.Wintrobes method

Sample collection :
In Westergrens method 2 ml of blood in 0.5 ml of 3.8% tri sodium citrate (1 in 4 parts
of blood) is taken.
In Wintrobes method 2 ml of blood in an EDTA vial is collected.

Westergrens method :
Westergrens pipette is 30 cm in length, with an internal diameter of 2.5 mm. Both ends
are open. Its marking are 0-200.

Procedure :
1. Mix well the anticoagulated blood sample
2. Fill the pipette with the help of rubber teat exactly upto zero mark.
3. Place the pipette upright in a stand with a spring clip at the top & rubber stopper at
the bottom.
4. Make it vertical using the adjustable leveling screws & allow it to stand for one hour.
5. After one hour plasma column above the column of red cells is read.

Precautions :
1. The pipette should be vertical as an angle of 30-60 from the vertical may accelerate
the ESR by as much as 30%.
2. Ratio of anticoagulant to blood should be 1:4.
3. There should not be any clot or air bubble in blood sample
4. The pipette must be kept in a vibration free bench.

Advantage : This is a more sensitive & accurate method as compared to Wintrobes

17
because the column is longer i.e. 200 mm.

Wintrobes method : Wintrobes tube is a glass tube, closed at one end and open at the
other. Its length is 11 cm & inner diameter is 2.5 mm. It has markings from 0 to 100 on
other side.

Procedure :
1. Fill the tube with the help of a Pasteur pipette. Tip of the Pasteur pipette is taken to
the bottom of the Wintrobes tube & blood is slowly filled into the tube by
withdrawing Pasteur pipette gradually.
2. Keep the tube in a vertical position for 1 hour in Wintrobes tube stand.
3. After one hour the plasma column height is taken from top mark of 0.

Advantages :
1. Simple method requiring small amount of blood.
2. After measuring ESR, same tube can be centrifuged to determine PCV.

Disadvantages :
1. Less sensitive as the column of blood is short.
2. ESR of more than 100 mm cannot be measured.

Normal values :
Westergrens Method:
Adult male < 15 mm in 1st hour
Adult Female < 20 mm in 1st hour

Wintrobes method :
Adult male < 7 mm in 1st hour
Adult Female <14 mm in 1st hour

Clinical significance of ESR :

1. It has prognostic significance.
2. Acute phase reactants like fibrinogen, CRP, 1 antitrypsin, ceruloplasmin are
elevated in acute infections resulting in an increase in ESR.
3. Chronic diseases like degenerative diseases & malignancy are associated with
alteration in plasma proteins & fibrinogen resulting in acceleration of ESR.
4. As a minor diagnostic criteria in case of Rheumatic fever.

Increased ESR :
Physiological :
1. Pregnancy
2. Menstruation
3. After exercise

18
Pathological :
1. Tuberculosis
2. Myocardial infarction
3. Anaemia
4. Rheumatic fever
5. Rheumatoid arthritis
6. SLE
7. Multiple myeloma
8. Walderstroms macroglobulinemia
9. Ankylosing spondylitis
10. Autoimmune hemolytic anaemia

Decreased ESR :
1. Polycythemia rubra vera
2. Sickle cell disease
3. Hereditary shperocytosis
4. Congestive Heart failure
5. Hypofibrinogenemia in liver disease.

Reference :
Todd- clinical diagnosis & management by laboratory method 17th edition.

19
Objective :To estimate Packed cell volume (PCV) or hematocrit of given blood sample.

Definition : PCV may be defined as the ratio of volume of packed RBCs to that of whole
blood which is expressed as percentage. PCV is estimated by two methods Macro &
Micro.

Collection of sample : 2 ml of blood in EDTA vial.

Methods of Estimation :
1.Macro Method
2.Micro Method
Macro Method :
1. Mix the blood sample & fill the Wintrobes tube by using Pasteur Pipette. Avoid
trapping of air bubbles.
2. Centrifuge the tube at 3000 rpm for 30 minutes.
3. After centrifugation, height of column of packed red blood cells is noted centrifuged
blood gets separated in 3 layers
4. RBC at the bottom
5. Middle layer of about 1 mm is Buffy coat
6. Topmost plasma layer.

Advantages :
1. The same tube filled for ESR can measure PCV.
2. The colour of plasma column may give additional information for example it is
yellow in jaundice, turbid in hyperlipidemia & red in cases of hemolytic anemia.
3. Buffy coat represents packed WBCs & platelets. Buffy coat thickness increases in
leukemia. It is also used for LE test.

Disadvantages :
If tube is not properly cleaned or dry then results may be erroneous other sources of error
are inadequate mixing of blood, excess anticoagulant trapping of leucocyte platelet clump
etc.

Micromethod :
Capillary tube is used in this method. Blood is centrifuged in a scaled capillary tube
which coated with anticoagulant. A special hematocist scale reader is used to read PCV.

Procedure:
1. of capillary tube is filled with blood obtained from finger prick or vein puncture.
2. Both ends are sealed with soft wax e.g. plasticize or by healing.
3. The sealed tube is centrifuged at 15,000 rpm from 3-5 minutes in high speed
centrifuge.
4. The height of the packed RBCs is taken against special hematocist reader.

20
Advantages :
1. Rapid, results are available within 5 minutes.
2. Less amount of blood is required.
3. More accurate.

Disadvantage :
Special centrifuge & hematocrit reader are required.

Normal value :
Adult male 40-54%
Adult female 38-47%

Clinical significance :

Increase in PCV is observed in:

1. Polycythamia Rubra Vera
2. Dehydration
3. Burn & shock

Decrease in hematocrit value is seen in :

1. Anemias
2. Excessive fluid volume of blood as in pregnancy.

Reference :
Todd clinical diagnosis & management by laboratory method, 17th Edition.

21
Objective :To prepare the peripheral smear and stain it.

Introduction : Peripheral blood film is a sheet of RBC with diffusely scattered

leucocytes and platelets and it provides information about RBCs, their number and
morphology (i.e. size,shape and other variations ), TLC,DLC and assessment of platelet
number and size.
Parts of peripheral smear:
Head, body & tail

Method of peripheral smear preparation-

Wedge method:
1.Slide on which the smear is to be prepared should be free from dust , scratches or oil.
2.The spreader should have smooth edges , be dry and the width should be less than the
glass slide on which the smear is prepared .If the edges are ragged ;tail of the smear is
formed which has majority of neutrophils .Rest of the smear shows paucity of neutrophils
,called as tailing of smear .
3.Slide is placed on a plain surface and held firmly with thumb and index finger ,put a
drop of blood not more than pin head size ,1cm away from the end.Hold the spreader
,place it at an angle of 45 degrees, in contact with drop of blood and make a smear with a
smooth, uninterrupted and forward movement of the spreader .Smear should be 2.5-
3.5cm in length .Air dry the smear.

Characteristics of good smear :

1.Surface should be even and uniform ,margins should not extend to the side of the slide.
2.Smear should be tongue shaped .
3.Film should not be too thin or too thick .

Thick smear : It is used for the detection of blood parasites e.g.malaria and microfilaria.

Procedure:
1.It is done by spreading a drop of blood on the slide in an area of about 2cm in diameter
and letting it dry .
2.This smear is given 2-4 dips in tap water till red coloured solution comes out
(dehemoglobinisation of the smear).
3.Let it dry and stain with the leishman stain .
4.If giemsa or field staining,then prior fixation with methanol is required .
5.Put a label with lead pencil at the head end of the smear.

Stains required:
Commonest Romanowsky stains which are made up of combinations of acid and basic
dyes are used .These are Leishman,Giemsa and Wrights stain.Romanowsky stains stain

22
red cells,platelets and parasites.

Constituents of Leishman Stain:

Leishman stain powder- 1.5 gm
Methanol (acetone free)-1000 ml

Composition of Leishman stain:

1.Basic dyes-Methylene blue.
2.Acidic dye-Eosin .
Nuclei (acidic ) will take up basic dyes and appear basophilic .Hemoglobin (being
basic) will take up acidic dye and appear acidophilic

Staining Procedure:
1 .Slide is kept on the rack.
2. Smear surface should face upward.
3. Pour Leishman stain and cover the whole slide with the stain.
4. After 1-2minutes,add buffer water (Sorensens phosphate buffer pH -6.8) or distilled
water on slide and mix buffer with the stain .
5.Let slide be stained by this mixture for 10 minutes .
6. Wash it with running tap water to remove excess of stain . Slide should be washed
from both the surface.
7. Keep the slide in vertical position for air drying .
8.. Label the smear
Buffered solution is used to maintain pH .If the pH is acidic, then red cells will stain pink
and WBCs stain very light and if the pH of the buffer is too basic, red cells will stain blue
and WBCs will take a bluish black hue.
Identification of proper mixing is that metallic sheen develops on the surface of film.
.

Examination of blood film:

1.Low power field examination Look for :
a.Quality of film
b.Number ,distribution and staining of WBCs .
c.RBCs examination Select an area where they just touch other without overlapping i.e.
between tail and body of film .
d Parasites
2.High power examination:
a.RBC Size, shape and Hb conc.
b.DLC
c.Platelets- number & morphology.
3.Oil immersion examination: DLC, parasites,platelet count.

23
Objective :To study the stained peripheral blood smear for Differential Leucocytic
Count.
Introduction :DLC is one of the important haemotological investigation performed
routinely in clinical practice. It helps in diagnosis of a disease and also studying its
prognosis.
White blood cells are divided into :
1.Granulocytes
a. Polymorphs / Neutrophils
b. Eosinophils
c. Basophils

2. Agranulocytes
a. Lymphocytes
b. Monocytes

Normal values of DLC :

1. Polymorphonuclear cells 40-80%
2. Lymphocytes 20-40% (Majority circulating lymphocytesare large lymphocyte &
10% are small.)
3. Eosinophils 1-6%
4. Basophils 1-2%

Absolute values :
1. Neutrophil 2000 to 7000 / mm3
2. Lymphocyte 1000 to 3000 / mm3
3. Eosinophil 20 to 500 / mm3
4. Basophil 20 100 / mm3
5. Monocyte 200 1000 / mm3

Differential leucocytic count is done on a blood film stained by Lishmans stain.

24
25
Comparative study of various white blood cells.
Granulocytes

Cells Size Nucleus Cytoplasm

(1) Polymorphs 10-15 m Stains deep purple & have 2-5 Cytoplasm contains numerous fine pink
lobes or violet granular
(2) Eosinophils 12-17 m Mostly bilobed Cytoplasm contains container coarse
dark purple granules
(3) Basophils 8-10 m Kidney shaped or bilobed Cytoplasm contain coarse dark purple
granules with usually obscure nucleus
Agranulocytes
(A) Lymphocytes
Small 7-8 m Large round nucleus Contain thin rim of cytoplasm
Large 12-15 m Nucleus may show small Cytoplasm is abundant clear & blue
indentation on one side
(B) Monocytes 15-18 m (Largest Large, curve after in shape of Bluish Gray cytoplasm with variable
granulative leucocytes) horse shoe number of fine reddish granules

26
Material required :
1. Peripheral blood smear stained with Leishmans stain
2. Cedar wood oil
3. Microscope

Procedure :
1. Examine blood smear under low power.
2. Put a drop of cedar oil & examine under oil immersion less.
3. Count the WBC in a zig-zag fashion across the breadth of the film in body & tail
junction area of film, until 100 leucocytes are counted. Counting should not be done
in body & tail area. It should be done in those areas where RBCs touch each other.
They should neither overlap nor widely spaced.
4. To record them draw a large square to divide it into 100 small squares. Identify one
cell & write in each square. Result obtained will directly give the DLC in percentage.

Clinical significance :

Neutrophilia :
1. Bacterial infections Staphlococcus, Streptococcus, Pneumococcus e.g. lobar
pneumonia, pyogenic meningitis, infected burns, Diphtheria.
2. Acute inflammatory conditions- Acute appendicitis
3. Acute stress states : Post surgery, Myocardial infarction.
4. Chronic myeloproliferative disorders like CML.

Neutropenia :
1. Infective disease like typhoid, paratyphoid
2. Physical agents - radiations
3. Haematological disorders Megaloblastic anemia, Aplastic anemia
4. Antimetabolite drugs like methotrexate

Eosinophilia :
1. Parasitic infections (Severe eosinophilia) eg. Filaria, round worm & hook worm
infestation.
2. Allergic conditions (moderate eosinophilia) eg. Bronchial Asthma, Urticaria
3. Hyper-eosinophilia syndrome (severe eosinophilia)
4. T & B cell lymphomas
5. Acute lymphoblastic leukemias.
6. Miscellaneous conditions like Tropical eosinophilia Loefflers syndrome.

Eosinopenia :
1. Typhoid
2. Prolonged use of steroids

27
Basophilia :
1. Myeloproliferative disorders.
2. Chronic myeloid leukemia (Basophils may be more than 10%)
3. Urticaria pigmentosa.

Lymphocytosis:
1. Chronic bacterial infections eg. T.B.
2. Viral infections Viral fever, Infectious mononucleosis, measles, chicken pox.
3. Chronic lymphocytic leukemia, NHL

Monocytosis :
1. Parasitic conditions Malaria & Kala azar
2. Chronic infections such as Tuberculosis, Sub acute bacterial endocarditis.
3. Inflammatory conditions such as Chrohns disease
4. Chronic Myelomonocytic Leukemia
5. Acute leukemias with monocytic component.

Arneth count
It is the count, in which neutrophils are counted according to number of lobes in their
nucleus. Normal mature neutrophil have five lobes, immature neutrophils have 1 or 2
lobes.
Shift to left : When number of lobes in the nucleus of neutrophil is less than normal i.e. 1
or 2, seen in Acute infections, Leukemoid reaction
Shift to right : When number of lobes in nucleus of neutrophils is more than five seen
Megaloblastic Anaemia & Uraemia.

Reference:
Practical Haematology by Sir John V. Dacie / S. M. LEWIS (Eight edition).

28
Objective :To estimate the total number of white blood cells per cubic millimeter of
blood .

Principle: Diluted whole blood is mixed with a fluid to lyse the red cells and stain the
white cell nuclei deep violet black. The diluting fluid used is Turks Fluid.

Methods of estimation:
1. Manual counting in Neubauers chamber
2. Automated cell counters

Apparatus and reagents required:

1. Turks fluid : Constituents of Turks fluid are
a. Glacial acetic acid -2 ml (Role of glacial acetic acid To lyse RBC)
b. Gentian violet -1ml (Role of gentian violet To stain the nucleus of WBC)
c. Distilled water -97 ml
2. Neubauers chamber

Procedure :
1. Using a WBC pipette, draw blood sample till 0.5 mark and clean the excess from the
tip.
2. Draw the WBC diluting fluid till 11 mark and mix.
3. Discard the initial 1-2 drops so that unmixed fluid filled in the stem is discarded and
only mixed blood with diluting fluid in bulb is used for counting.
4. Place the clean coverslip on the clean and dry Neubauers chamber and charge the
chamber.
5. To charge the chamber, the pipette is slightly inclined to the chamber and the pressure
is released gently with the finger to allow a small volume of fluid to flow down. The
fluid will be contracted under the coverslip by capillary action. Allow the cells to
settle down for 1 min.
6. Examine under the microscope and then count the number of cells in the WBC
squares in high power(40x).

Improved Neubauers chamber :

Identification :
The improved Neubauers chamber has a metallized surface. It has two ruled
stages separated by a small gutter. The two stages in turn are separated from two ridges
by gutters, one on each side .
Ruled area :
The ruled area is of 3x3 mm on each chamber stage. This is divided into 9
squares of 1x1cm each. The four big corner squares measure 1x1 mm each and each of
these is divided into 16 small squares .These are used for counting white cells .
The central 1x1 mm square is divided into 25 squares, each of which is in turn
divided into 16 small squares. The area of each 25 small square measures 1/5 x1/5 mm.
Five of these medium squares i.e. the 4 corner squares and 1 central square are used for

29
estimating the total red cell count .
The depth of the the grooved area is 0.1 mm, so the total volume of each big
square is 1x1x0.1mm =0.1 cumm and the volume of the middle square in the central area
is 1/5 x 1/5 x 1/10 mm =1/250 cumm .
Coverslip :
The cover slip should have very smooth surface and even thickness (0.3 mm ,0.4 mm
or 0.5 mm ). The 0.4 mm thick coverslip is most commonly used.

Calculation :
TLC /cumm = No of WBC counted (N) x Dilution (D)
Area counted x depth (V)
N = number of cell counted in the 4 corner squares
D = 20
Area counted = 4x1x1 sq mm = 4 sqmm .
Depth = 0.1 mm
V = Area x depth
TLC = N x 20 / 4 x 0.1 = N x 50 cells /cumm.

Precautions :
1 Major sources of error are improper volume measurement, improper charging of
chamber, use of defective pipette, improper counting, failure to clean WBC pipette,
Neubauers chamber and cover slip and wrong calculation .
2 Once the chamber is filled, counting should be done as early as possible before the fluid
dries up and air bubbles enter the chamber.
3 Use clean and dry glassware(including pipette, chamber and cover slip).

Uses of Neubauers chamber:

1. TLC
2. TLC of body fluids including CSF
3. Sperm count
4. Platelet count
5. RBC count

30
Normal value: Adults : 73 x 1000 /cumm 1
At birth : 188 x 1000/cumm1
Children(up to 6 years ) :10 5x1000 cells /cumm1

Causes of leucocytosis :
1. New born, pregnancy, exercise, emotional stress, acute infection, leukemia .
2. Causes of Leucopenia
3. Typhoid , Megaloblastic anaemia ,bone marrow depression.

References:
Dacie and Lewis Practical Hematology; 9th edition: (pg 12-13)

31
Objective :To perform the Total Red Blood cell count in the given blood sample.

Introduction : Red blood cell count is itself important in diagnostic haematology and
also because it permits the calculation of Mean Cell Volume (MCV) and Mean Cell
Haemoglobin (MCH).

Methods of RBC Count :

1. RBC count by Manual Method
2. RBC count by Electronic cell counter.

Manual Method for RBC count :

Material Required :
1.RBC Pipette
2.Neubauers chamber
3.RBC diluting fluid
4.Microscope
5.Blood sample

RBC Diluting Fluid : The various RBC diluting fluids are :

1.1% Formal Citrate is used as RBC diluent in our laboratory Contains 40%
Formalin, Trisodium ciltrate, Distilled water.
2.Toissons Fluid Contain Glycerine, Sodium Sulfate, Sodium Chloride, Methyl
Violet & Distilled water.
3.Hyems fluid Contains Sodium chloride, Sodium sulfate, Mercuric chloride &
Distilled water.

Precautions for using diluent :

1.Diluting fluid should be isotonic, so that RBCs do not haemolyse.
2.Do not use normal saline as diluent as it causes crenation of RBC :

Procedure :
1. RBC count can be done either directly on capillary blood or venous anticoagulated
blood is used.
2. Fill RBC Pipette upto mark 0.5 with blood, wipe the pipette from outside.
3. Draw immediately RBC diluting fluid upto mark 101 of the RBC pipette & rotate to
mix. Now the dilution is 1:200. (Dilution factor 200).
4. Discard the first 1-3 drops from the RBC pipette.
5. Place cover slip over dry & clean Neubauers chamber in such a way that it covers
both ruled platforms completely.
6. Charge the chamber in one action with cover slip on it.
7. Allow cells to settle for 2 minutes.
8. Count RBC in the centre of the chamber (Four corner square & one centre one).

32
Precautions while changing chamber.
1. Neubauers chamber should be clean & dry.
2. Charge the chamber in one action
3. Leave chamber for two minutes after charging, so that RBC may settle but not more
than this as there may be drying at the corner.
4. Chamber should be charged completely.
5. There should be no air bubbles under the coverslip in the chamber.
6. There should not be any vibration on the working bench.

Calculations :
Volume of one RBC square Length x Breadth x Depth
=1/5x1/5x1/10 = 1/250 cub mm
Volume of five RBC squares = 5x1/250 = 1/50 cub mm
Supposing no of RBC in 5 squares = N
Dilution factor 1 in 200.
Hence actual no. of RBC = (Nx200)/W = Nx50x200
= Nx10,000 RBC /cub
Results of given sample : Actual no. of RBC = RBC/ mm3.

Normal RBC count :

At Birth 6.0 + 1.0 million/cub mm
2-6 years 4.6 + 0.7 million/cub mm
6-12 years 4.6 + 0.6 million/cub mm
Adult female 4.3 + 0.5 million/cub mm
Adult male 5.0 + 0.5 million/cub mm

Clinical significance :
Increased RBC count :
1. Haemoconcenteration due to burns, acute gastroenteritis etc.
2. Central cyanotic stages eq. congenital heart disease, emphysema
3. Porphyria,
4. Polycythaemia.
Decreased RBC count :
1. Anaemia
2. Haemodilution
3. Old Age

33
Objective :To perform the Platelet count of given sample of blood.
Procedure :
1. Manual method.
2. Electronic counter method.

Material Required :
1. Diluent
2. Neubauers chamber
3. WBC pipette/ RBC pipette
4. Blood sample
5. Microscope
Diluent : 1% Amm. Oxalate solution (Ammonium oxalate & Fresh Distilled water) is
used. It haemolyses the blood cells except platelets.

Method :
1. Fill WBC pipette upto mark 0.5 with blood, wipe the pipette
2. Now fill the pipette upto mark 11 by 1% Amm. Oxalate diluent.
3. Mix well by rotating the pipette
4. Discard 1-2 drops.
5. Charge the Neubauers chamber.
6. Keep on moist filter paper in a petridish for 5 minutes, so that platelets settle down.
7. Count platelets in central 1x1 sq. mm.

Calculation : No. of platelets - n

Dilution - 20
=N x Dilution/ vol.
Area 1x1x1/10 mm3.
Platelet count = (Nx20)/V = Nx20
1/10
= nx20x1x1/10 = nx200 cub mm

Normal Range : 1.5-4 lacs platelets/cu mm

Precautions :
1. Reduce illumination by closing diaphragm
2. Lower condenser, platelets can be as seen highly refractile particles.
3. Sources of error :
4. Discard and repeat if platelets are agglutinated.
5. Always review platelets in a stained smear.
Technical errors :
1. Poor collection of blood sample
2. Insufficient mixing
3. Faulty charging of chamber
4. Presence of dust particles

34
Clinical significance:

Thrombocytopenia :
When platelets are less than 1 lac / cub mm.
1. Acute Idiopathic thrombocytopenic Purpura
2. DIC
3. Drug induced Chloramphenicol toxicity
4. Pancytopenia Aplastic anemia, Hyperspleenism. Megaloblastic anemia, Acute
leukemia.
5. Radiation.

Thrombocytosis :
1. When platelet count is more than 4 lac/ cub mm
2. Idiopathic Thrombocythemia
3. CML
4. Primary proliferative polycythacemia
5. Post haemorrhage
6. After Burns.

35
Objective :To study and identify the blood group.

Introduction:
Blood banking : It is relatively newer branch of medicine.Modern blood banks have
multiple activities.:
1.Blood donation
2.Testing of blood for transfusion transmitted diseases.
3.Blood serology including blood grouping,crossmatching,antigen and antibody detection
4.Blood component preparation.
5.Blood storage
6. Consultation for requirement of components
7. Resolving transfusion reactions.
8. Apheresis component collection
9.Therapeutic plasma exchange
10. Stem cell collection & storage
11 Other organs storage
Whole blood can be divided into several components like packed red blood cells,fresh
frozen plasma,platelets concentration,cryopoor plasma etc.

Apheresis: This is a procedure done through an automated machine in which blood

comes to machine ,specific component is separated & rest of the blood enters back to the
donor body. In this procedure,increased amount of required component can be produced
without harming the donor as compared to whole blood donation.
Same machine can be used for the collection of haematopoitic stem cells required for
transplantation.

Existence of blood group was discovered by Landsteiner in 1900 .There are more than
29 blood group systems .
Human RBCs contain on their surface a series of glycolipids and glycoprotiens which
constitute a blood group antigen .

The clinically important blood group systems are A BO and Rh systems .Other minor
systems are MNS ,P ,Kell ,Lewis and Duffy etc .
Human RBCs contain on their surface a series of glycolipids and glycoprotiens which
constitute a blood group antigen .

ABO system :

ABO is the most important blood group system because Anti A and Anti B are found as
naturally occurring antibodies in virtually all subjects who lack the corresponding
antigens and these antibodies often cause intravascular haemolysis when incompatible
RBC are transfused. There are 4 major groups in A BO system . A,B, AB and O which are
determined by presence or absence of A/B antigen .

Two main antigens A,B result in blood groups A,B,AB and O . These antigens are

36
under the control of A and B genes .Gene H governs the expression of gene A and gene
B .Most individuals are homozygous for gene ( HH)

Bombay phenotype :They are individuals who lack H gene (genotype hh) Therefore no
H substance is formed and no A/B antigen develops even though individual may possess
A/B genes .They are tested as group O and possess anti A ,anti B and anti H antibodies
Bombay blood group .The Oh phenotype becomes apparent when serum of Oh person is
tested against group O red cells and strong agglutination or haemolysis occurs, therefore
O blood group cannot be transfused to Oh individuals

Antigens & antibodies in the ABO system:

Group Antigens on red cells Antibodies in serum

A A Anti B
B B Anti A
AB A, B -
O - Anti A Anti B

Rh system :Rh antigen was first discovered on red cells of Rhesus monkey . .Rh antigen
is present in 85 % of human beings are Rh + ve and those who lack the antigen are Rh
ve .

Grouping techniques:

Slide technique : For ABO grouping and Rh grouping .

1.Take a slide and mark A,B.Take another slide and mark D
2..Add 1drop of washed 10 % red cell suspension ( or according to reagent manufacturer)
and put 1 drop each of antiserum (A,B and D )
3.Mix each separately and read for agglutination macroscopically within 5min and
identify the blood group as well as Rh group .
4.Agglutination in anti-D indicates Rh +ve blood sample and absence of agglutination in
anti-D indicates Rh-ve blood group .

Antibody added AntiA AntiB AntiD Result group

Agglutination - - + O +ve
+ - + A +ve
- + + B+ve
+ + + AB +ve

Anti-sera A is blue in colour

Anti-sera B is yellow in colour
Anti-sera D is colourless

37
Tube method for ABO grouping and Rh grouping:

Forward grouping:
1.Take 3 test tubes and label it as A ,Band D.
2.Add 1 drop of corresponding antisera to each tube that is Anti-A , Anti-B and Anti-D .
3.Add 1drop of 2% red cell suspension to 3 test tubes and mix the suspension by gently
tapping the tubes , incubate at 37 degree celsius for 45 minutes and then centrifuge it at
1000 rpm for 1 minute.
4. Look for agglutination macroscopically as well as microscopically and interpret the
result for blood group ..

Reverse grouping :
1.Take 2 test tubes and label it as test tube A and test tube B .
2. Add 2 drops of unknown sera of patient in tubes labeled as A and B .
3.Add 1 drop of 5% red cell suspension of known blood group i.e. A and B .
4.Centrifuge at 1500 rpm for 1 min .
5 Look for agglutination .

Unknown sera of patient + known A cell Sera+ known B cell Result

- - AB
- + A
+ - B
+ + O

Blood group distribution :

Blood group Indian population
A 25%
B 37%
AB 5%
O 33%

Importance of blood grouping :

1. Blood transfusion It is mandatory prior to transfusion in any individual.

2. Hemolytic disease of newborn .
3. In cases of paternity disputes .
4. Medicolegal purposes .
5. Usefulness of blood grouping in immunology , genetics and anthropology.

Cross matching :
It is the most important test carried out before blood transfusion .This is carried out to
prevent transfusion reactions by detecting antibodies in the recipients serum that may
react with donors red blood cells .

38
Routine cross matching :

Major cross match :

One of the simplest method is :
1.Take 1 drop of recipients sera in a small test tube .
2.Add 2- 5% saline suspension of washed donors red cells to the recipients sera . 3.Mix
the two .
4.Incubate at 37degree cel for 45 minutes.
5.Centrifuge at 1000 rpm for 1min .
6.Dislodge the cells gently and examine macroscopically and microscopically for
agglutination or lysis . There is no agglutination or hemolysis in matched recipients and
donors samples . In mismatching of donor and recipient , hemolysis / agglutination
occurs .

Minor cross match:

Recepients RBC is matched with donors sera in the same fashion as above.

Transfusion reactions :
They are divided into 2 types :

Acute transfusion reaction :

1.Haemolytic
2. Non hemolytic :
a. Febrile
b. Allergic
c. Anaphylactic reactions .
d. Non cardiogenic pulmonary reactions known as transfusion related acute lung
injury (TRALI)
e. Bacterial sepsis
f. Volume overload

Delayed transfusion reaction :

1. Hemolytic reaction
2. Transfusion related graft vs host reaction
3. Post transfusion purpura
4. Post transfusion hemosiderosis
5. Blood transmissible diseases like hepatitis B and C ,HIV , CMV , Malaria ,Syphilis
etc.

39
Objective : To study the various types of anaemias

Definition: Anaemia is present when the haemoglobin level in the blood is below the
lower extreme of the normal range for the age and sex of the individual (Men- 13-
17gm/dL, Women- 11-15gm/dL)

Microcytic Hypochromic Anaemia (IDA):

Causes:
1. Iron deficiency anaemia ( IDA )
2. Thalassemia major
3. Anaemia of chronic disease

Routine investigations:
1. Hb - decreased
2. TLC- normal
3. Platelet count - Normal/Increased
4. MCV< 80fl (Normal range- 85-95fL)
5. MCHC<30g/dl ( Normal range- 31-34gm/dL)
6. MCH<27pg ( Normal range- 27-32pg )
Special investigations:
1. Serum Fe<10umol/l (13-32 umol/l)
2. Serum ferritin<12ug/l ( men-20-300ug/l, women- 15-150ug/l)
3. Marrow Fe-decreased/absent
4. TIBC Increased (2.5-4mg/l)

Peripheral blood smear findings:

Red Blood cells:
1. Smaller than normal.
2. Central pallor is more than half of the red cell diameter.
3. Mild to moderate degree of anisopoikilocytosis with microcytosis.
4. Ring / Pessary cells In severe hypochromia ,central pallor becomes 2/3rd to 3/4th
of the cell .Cells reveal only a thin rim of hemoglobin at the periphery .
5. Few tear drop cells and elliptical cells are also present.
DLC:
Normal except in cases of hookworm infestation (may be associated with eosinophilia)

Bone marrow findings:

1. Bone marrow is hypercellular
2. Erythroid hyperplasia is present
3. Micronormoblastic reaction is present
4. Myelopoiesis & megakaryopoiesis are normal.
5. Bone marrow iron is depleted. ( Special stain used to demonstrate iron- Prussian
Blue / Perls stain)

40
Macrocytic Anemia

Causes:
1. Vitamin B12 / folate deficiency
2. Liver disease
3. Alcohol excess.
4. Hypothyroidism

Routine Investigations:
1. Hb Decreased (moderate-severe)
2. TLC-Normal / Decreased
3. Platelet count- Normal / Decreased
4. Severe macrocytic anemia may be associated with pancytopenia
5. MCV>100fl
6. MCHC-Normal
7. MCH>35pg
Special investigations:
1. Serum B12 levels
2. Serum and Red cell folate
3. Schilling test
4. Intrinsic Factor antibodies
5. Thyroid hormone levels

Peripheral blood smear:

Red cells:
1. Show mild to moderate degree of anisopoikilocytosis.
2. Macrocytes, macroovalocytic red cells are noted.
3. Presence of tear drop cells,elongated cells and red cells with inclusions like Howell
jolly bodies (nuclear remnant).
4. Basophilic stippling and Cabot rings.
5. Occasional late megloblast may be seen.
WBC:
Mcropolycytes with the presence of Hypersegmented neutrophils (Neutrophils > or = 6
lobes).
Platelets:
Normal or decreased .

Bone Marrow findings:

1. Megaloblastic erythroid hyperplasia.
2. M: E ratio - 1:1-1:6 (normal 3-7:1) .
3. Dyserythropoiesis with increased mitosis is seen.
4. There is presence of giant myelocytes and metamyelocytes.
5. Megaloblasts are larger than normoblasts and have open sieve-like nuclear chromatin.

Normocytic normochromic anaemia:

41
Anaemia with normal red cell indices

Causes:
1. Post haemorrhage anaemia
2. Renal diseases
3.Marrow suppression

42
Objective :To study the blood picture of leukemia.

Definition:They are malignant neoplasm of blood forming cells which undergo

uncontrolled proliferation in bone marrow.

Classification:
Acute- 1. Myeloblastic
2.Lymphoblastic
Chronic-1.Chronic Myeloid leukemia
2.Chronic lymphocytic leukemia

Acute leukemia:
1. They are characterized by predominance of undifferentiated leucocyte precursors or
leukemic blasts.
2. A leukemia is acute if the bone marrow consists of more than 20% blasts.(WHO)
3. Diagnosis of acute leukemia is made by a combination of routine blood picture and
bone marrow examination , coupled with cytochemical stains and certain biochemical
investigations.

Peripheral blood smear findings of acute leukemia:

1. Anaemia Normocytic Normochromic type

2. Leukocyte count- Variably elevated and usually it is 1 lakh/cumm.However it can be

less than 10,000 in some cases where it is called Subleukemic leukemia. Majority of
the cells are monotonous blast cells containing large vesicular nuclei and small
amount of cytoplasm.

3. Platelet count-Almost always there is thrombocytopenia ranging from moderate to

severe.

AML

Morphologic characteristics of blast cells:

1. Size-10-18um
2. Nucleus Round or oval
3. Nuclear chromatin-Fine
4. Cytoplasm-Scanty to moderate with fine azurophilic granules,Auer rods may be seen.
5. Nucleoli-3-5
6. Cytochemical stain-Peroxidase stain +ve except in Mo type / Sudan black positive

ALL

43
Morphological characteristics of blast cells:
1. Size -10-18um
2. Nucleus Round or oval
3. Nuclear chromatin-Slightly clumped(coarse)
4. Nuclear membrane-Fairly dense
5. Nucleoli-1-2
6. Cytoplasm-Scanty,clear blue,agranular.
7. Cytochemical stain- PAS(Periodic Acid Schiff) stain +ve
`
Prognosis: ALL has good prognosis as compared to AML

References:
1. De Gruchys Clinical Haematology in medical practice
2. Robbins pathologic basis of disease.

CML : Seen in 4th decade of life.

Both
CLL :
Malignancy of mature lymphocytes in elderly.
Disease of elderly (over 60 years)
Male : female = 1:2
Routine investigation
PBF
Anemai mild moderate
Normocytic normocheovice
Account for 25T of all cases of leukemia in U.S.
Malignancy of mature B lymphocytes in elderly
Age group : Occurs in persons over 50 years. Males are affected twice as commonly as
female
Clinical features :
Patient presents with generalized lymphadenopathy
Hepato spleenomegaly
Other non-specific symphoms easy faty ability 100 of
PS leucocytosis may reach

44
Objective :To study the bleeding time of the given sample.

Definition : Bleeding time is the measure of platelet number and platelet function;
capillary function and platelet plug formation.

Principle: A standard incision/ prick is made on the volar surface of the forearm/tip of
finger or ear lobe (depending on the specific method used), and the time during which it
bleeds is measured and the cessation of bleeding indicates the formation of platelet plug.
This is dependent on an adequate number of platelets and the ability of the platelets to
adhere to the subendothelium and to form aggregates.

Methods of estimation:
1. Dukes method
2. Ivys method
3. Template method

Apparatus required:
1. Filter paper
2. Pricking needle/ lancet (depending on the method)
3. Cotton, spirit
4. Stop watch
5. Sphygmomanometer (for Ivy & template methods)

Dukes Method

Procedure:
1. Clean the tip of finger/ ear lobe with a spirit swab.
2. A bold prick is given to the tip of the left ring finger or the lobe of the ear and the stop

45
watch started.
3. The blood is dropped upon the filter paper every 15 seconds till the bleeding stops.
4. Do not squeeze the finger/ ear lobe.
5. When the bleeding stops, the wound is touched gently with a filter paper at 15-30 sec
interval.
6.When the blood no longer stains the filter paper, the watch is stopped and the time
noted.

Normal range : 6-10 min

Ivys Method:

Procedure:
1. A sphygmomanometer cuff is fixed upon the patients arm above the elbow and
inflated to 40 mm of Hg and the same pressure is maintained throughout the test.
2. The volar surface of forearm is cleaned with rectified spirit and an area of skin devoid
of superficial veins is chosen.
3. The skin is stretched laterally between the thumb and forefinger of the left hand and
two separate punctures 5-10 cm apart are made in quick succession by a free hand; using
a disposable lancet /surgical blade (cutting depth of 2.5 and width of 1 mm)
4. The timer is started as soon as the puncture is made and the bleeding starts.
5. Blood exuding from the cut is blotted out gently but completely with a filter paper; at
15 or 30 sec interval; till bleeding stops.
6. The time is noted .
7. When bleeding has ceased, a sterile adhesive strip is applied on the wound .

Normal range: 2-7 min

Template Method

Procedure:
1. Firstly the forearm is cleaned with alcohol and air dried.
2. Sphygmomanometer cuff is inflated upto 40 mm of Hg at the upper arm.
3. Place the template 5 cm distal to antecubital crease, avoiding superficial veins.
(The template is made up of metal /plastic and has a longitudinal slit of 6 mm length and
the thickness of plastic and the angle of the blade is such that the incision made through
the slit is 6 mm long and 1 mm deep).
4. The template is pressed against the flattened skin surface and the slit made is parallel to
antecubital crease.
5. The blade is introduced into the slit and the skin is penetrated and the incision made
with a smooth rapid movement along the entire length of the slit.
6. A stop watch is started immediately after the incision is made.
7. The skin incision is blotted with filter paper without touching the edges of skin every
15 seconds until blood stops flowing.
8. The time taken for cessation of blood flow is noted as the bleeding time of the patient.
9. Now the edges of the skin are apposed by applying adhesive tape.

46
Note : According to the modified Dukes method, another incision is made 1.5-2 cm away
from the first one and then another stop watch is started.. Mean of the 2 readings is taken
as the bleeding time of the patient.

Normal Range: 2.5-9.5 min.

Causes of prolonged bleeding time:

1. Thrombocytopenia
2. Platelet function disoders Thrombasthenia, drugs.
3. Von Willebrands disease
4. Vascular abnormalities Pseudoxanthoma elasticum ,Ehler Danlos Syndrome.
5. Uremic Patients
6. Antiplatelet medication-eg. aspirin

Objective :To study the clotting time of the given sample

Definition: The time taken by the shedded blood to form a clot is known as clotting time.
C.T. indicates the efficacy of the intrinsic pathway of coagulation and is prolonged only
when there is severe deficiency of factor VIII, XI or fibrinogen and in heparin therapy .

Principle: Venous blood is withdrawn from the patient and the time taken by this blood
to clot is noted as the clotting time.

Methods of estimation:
1. Capillary tube method of Wright
2. Lee and White method

Apparatus required:
Material for collection of blood:
1. Cleansing swabs
2. Stop watch
3. Capillary glass tubes (for the capillary tube method)
4. Test tubes (10x1cm size) x 3 (for Lee and White method)
5. Water bath at 37C

Capillary Tube Method Of Wright:

Procedure:
1. After aseptic precautions, a bold prick is given on the left ring finger and blood is
drawn into the capillary glass tube.
2. The time of appearance of blood on the ring finger is noted and the stop watch
started.
3. The capillary glass tube is then kept between both the hands for one minute and then

47
 the tube is taken and small portions of the tube are broken at regular intervals of 15
seconds until a thread of clotted blood appears between the two pieces of the tube .
4. The time interval between the appearance of the blood and formation of thread is
noted.

Lee And White Method:

Procedure:
1. Withdraw 3 ml of venous blood using aseptic precautions.
2. Start the stop watch, the moment blood appears in the glass syringe.
3. Deliver 1 ml of blood each into 3 small test tubes (10 cm by 1 cm size) labeled as 1,2
& 3, already placed in a water bath at 37 Celsius.
4. After 3 minutes, tube no 1 is taken out and tilted to 90 degree to look for clot formation
and the same procedure is repeated every 30 secs .
5. The time when the tube can be inverted without its contents spilled out is noted.
6. This is repeated with the other two tubes also and the average of the C.T. of the three
tubes is taken, giving the final result .

Normal Range: 6 17 mins (glass tubes)

19 - 60 mins (siliconozed tubes)

Causes of prolonged CT:

1. Haemophilia
2. Obstructive jaundice.
3. Liver diseases due to deficient prothrombin and fibrinogen formation.
4. New born babies due to low prothrombin levels during early days of life.

Causes of shortened CT:

1. After prolonged haemorrhage.
2. After blood transfusion.
3. After antibiotic treatment.
4. Febrile state

Precautions:
1. The prick should be bold so that blood flows freely.
2. Size of the tube should be uniform and preferably 1 cm in diameter and 3 cm in
length.
3. Tube should be totally clean, since any substance coating the inner surface will affect
C.T.
4. When C.T. is markedly prolonged, as in haemophilia , it may take even up to 1 hr for
clotting to occur .

References:
1.Dacie and Lewis Practical Hematology; 9th edition; pg 12
2.Interpretation of Diagnostic tests by Jacques Wallach, MD; 8th edition; pg 12

48
Introduction:
Urine examination is important for diagnosis of various diseases. Complete routine
examination is divided into four parts-
1. Collection of urine
2. Physical examination
3. Chemical examination
4. Microscopic examination

Objective :To perform the physical examination of urine.

Collection of Samples:
1. Early morning sample
2. The patient is asked to bring the urine which he voids first in the morning in a clean
bottle without any contamination .Early morning samples are preferred since these
have the lowest pH which preserves the formed elements well and are the
concentrated samples therefore casts and crystals etc if present are easily
demonstrable .
3. 24 hr collection of urine for quantitative tests The patient is asked to empty his
bladder at 8 a.m. or a suitable time and to discard the urine . He collects all
subsequent urine upto and including that at 8 a.m. the following morning . The total
volume of this sample is measured and recorded and urine mixed before analysis.
4. Mid stream sample preferred for microbiological examination.
5. Random samples for qualitative tests .

Other methods of collection are :

1.Suprapubic catheterization
2.Catheterization
Specimen bottles should be labeled clearly

Preservation of sample :
It is done to prevent growth of contaminant .This can be done by:
1. Refrigeration at 4 degree celsius
2. Toluene (1ml/50ml of urine) - it forms surface coating over urine and is best all-
round.
3. Thymol crystals may be kept in container (0.1gm/100ml)
4. Chloroform
5. Boric acid- 5gm/4 oz of urine
6. Formalin-6-8drops of 40% formalin/100ml urine
7. Conc. HCl (10ml /24hrs )

Routine examination of urine : Routine specimens should be examined within 1 to 2

hours after voiding.

49
Physical examination of urine :
1.Volume
2. Turbidity/appearance
3. Colour
4 Odour
5. pH
6. Specific gravity

1. Volume: 1.2 -1.5 l/ 24 hrs in normal individuals .

a . Polyuria : is a condition in which urine output is increased more than 2.5 l /24 hrs .
Physiological causes :
1.Cold weather
2.High intake of fluids
Pathological causes:
1. Diabetes mellitus .
2. Diabetes insipidus .
3. Patient on diuresis .
4 .Nephritis
5. Patient on psychosomatic drugs .

b. Oliguria : Urine output is decreased to less than 500 ml / 24 hrs .

Causes :
Physiologic :
1.Hot weather
2.Reduced fluid intake
3.After exercise
Pathological:
1.Fever
2.Diarrhoea
3.Chronic glomerulonephritis (early stage)
4.Acute liver condition
5. Cardiac failure

Anuria: When quantity is markedly diminished less than 100 ml/ 24 hrs
Causes:
1.Shock
2. Stricture urethra, retention of urine as in Benign Hyperplasia of prostate
3. Stones ,tumours in kidney
4. Acute nephritis
5. Mercury poisoning
6.Incompatible blood transfusion

2..Appearance :
Normal urine is clear but may be cloudy due to presence of:
a. Amorphous phosphates in neutral or alkaline urine

50
b. Amorphous urates in acidic urine
c. Turbid due to pus
d. Bacteria and fungi
e.Fat and chyle

3. Colour: Colour of normal urine is straw or amber coloured. It is due to urochrome and
urobilin. Light or dark coloured urine may be seen in increased or decreased intake of
fluid respectively.Some of the e.g.of pathological changes in colour-
1. Deep amber-after exercise,high grade fever.
2. Reddish brown- increased urobilinogen or porphyrins
3. Bright red- Large amount of fresh blood
4. Pink- Small amount of fresh blood
5. Smoky brown-Blood pigments
6. Brownish yellow or green with a yellow foam-Bile pigments-jaundice
7. Milky white- chyluria due to filariasis
8. Orange due to drugs like rifampcin
9. Green-phenol poisoning

4..Odour : Normal odour is aromatic .

1. Odourless-Acute renal failure
2. Ammoniacal-If allowed to stand at room temperature for sometime.
3. Mousy odour-Phenylketonuria
4. Fruity odour-Ketone bodies present
5. Pungent- due to ammonia produced by bacterial contamination
6. Putrid- due to urinary tract infection

6. pH:
Normal urine pH ranges from 4.7-7.0.Constituents responsible for acidity are pyruvic
acid ,lactic acid ,citric acid, ketone bodies . Acidic pH in high protein diet & alkaline
pH in vegetarian diet .

pH is measured :
1.Litmus paper Acidic urine blue turns red
Alkaline urine red turns blue
2.pH indicator paper
3.Strip multistix method .

Acidic urine:
1.High protein diet e.g. meat
2. Ingestion of acidic fruits
3.Respiratory and metabolic acidosis
4. U.T.I. by E.Coli.

Alkaline urine :
1.Respiratory and metabolic alkalosis.
2.U.T.I. by Proteus,Pseudomonas

51
6.Specific gravity :
Ratio of weight of 1ml volume of urine to that of weight of 1ml of distilled water.
Specific gravity is used to measure the concentrating and diluting power of the kidneys.
Normal specific gravity of a 24hrs urine sample is 1.003-1.030 .

Methods of measuring Specific Gravity :

1.Urinometer
2.Refractometer
3.Dipstick method
Urinometer and Refractometer methods are more accurate as compared to dipstick
method.

Urinometer:
Procedure:
1.Fill urinometer container with urine .
2 Float urinometer into it so that it floats in urine without touching the wall and
bottom of container
3. Read the graduation on the arm of urinometer at lower urinary meniscus.
4. Add or substract 0.001 from initial reading for each 3 degree celsius above or
below the calibration respectively marked on the urinometer.

Specific gravity is increased in:

1. Dehydration
2. Restricted fluid intake
3. Diarrhoea
4. Vomiting
5. Fever
6. Excessive sweating
7. Diabetes mellitus
8. Albuminuria
9. Acute glomerulonephritis .

Specific Gravity is decreased in:

1. Excessive fluid intake
2. Diabetes insipidus
3. End stage kidney chronic glomerulonephritis and other renal diseases like bilateral
polycystic kidneys , chronic pyelonephritis and hypertension
4. When concentration power of kidney is severely affected it results in a low and fixed
specific gravity of 1.008 -1.010.eg ESKD ,Renal failure .

Urine concentration and dilution test :

This test assesses the ability of kidneys to concentrate urine . The patient takes fluid or
restricts fluids but irrespective of it ,the specific gravity of urine is low and fixed at 1.010
indicating the total loss of concentrating power of kidney .

52
Objective :To perform the chemical examination of urine.

Chemical constituents frequently tested in urine are-

1. Protein
2. Glucose
3. Ketones
4. Blood
5. Bile derivatives

1. Protein : Any detectable amount of protein in urine is abnormal .Less than 150 mg /
day is normal protein in urine .

Causes of proteinuria :
A. Organic proteinuria :

Heavy: More than 3-4gms / day . It is found in:

1. Nephrotic syndrome
2. Acute or chronic glomerulonephritis
3. Toxemia of pregnancy
4. Heavy metal poisoning
5. Amyloidosis of kidney
6. Diabetic nephropathy

Moderate: 1-3 gms / day . In all the above conditions and toxic nephropathy ,
pyelonephritis , CHF .

Minimal : Less than 1 gms / day . Found in:

1.CHF
2.Renal TB
3.Pyelonephritis .

B. Accidental proteinuria : Test will be +ve , if contaiminated by blood, mucus or

semen, vaginal discharge.

C. Functional proteinuria: It is found after severe exercise , high protein diet and during
late stages of pregnancy, after prolonged cold bath.

D. Postural proteinuria: Erect posture causes proteinuria but is absent in recumbent

position .Probably because of unusual anatomical arrangement of kidney vessels which
are compressed when the body is upright . Proteinuria is absent in morning sample and
present after 2hrs of mobility .

E. Bence jones proteins: These are abnormal protein found in urine in

multiple myeloma and macroglobulinemia. These proteins appear at 40-60

53
degree celsius and then become soluble again at 100 degree celsius .
If both albumin and Bence jones protein are present , we first boil the sample to
precipitate albumin , then filter it .Bence jones protein comes in filtrate . Now we cool
the filtrate at 40-60 degree celsius and see for Bence jones protein which will appear on
cooling.

Chemical tests for protein:

Heat and acetic acid test:

1.Fill 3/4th of a preferably small diameter but long test tube with urine
2. Boil the upper or 1 inch of the column of urine . If proteins or phosphates or
carbonates are present a white cloud appears in the heated portion .
3.We heat the upper layer as lower layers serve as a comparator control
4. Add 2-3drops of 3% acetic acid , if cloudiness disappears on addition of acetic acid ,
then it is due to phosphates and if still persists ,it is due to proteins .

Grading of proteins :
a. If the protein is present in trace (<0.1gm%) Just barely visible cloudiness
b. + (>0.01gm%) Slight turbidity and no flocculation .
c. ++ ( 0.1- 0.2gm%)- Dense turbidity and no flocculation.
d. +++ (0.2-0.4gm%) Dense turbidity and granular flocculation
e. ++++(0.5gm%) Curdy white ppt .

Multistix:
Albustix ,combistix ,labistix etc are now available in India and make testing for
albumin much simple and quicker .These are all based on the same principle and contain
an indicatorbromophenol.If albumin is present the deposit must be examined for urinary
casts /pus cells .

Other tests are sulphosalicylate acid test in which we take 3-4 ml urine, add 2-3 drops
of 3% sulphosalicylic acid. Appearance of precipitate within 5 min. denotes presence of
protein.

Quantitative estimation of protein in urine:

1.Quantitative estimation of urine is done by Esbachs albuminometer.
2.It is shaped like a test tube and is graduated in grams of dried albumin/1000ml of
urine.U and R are present on the tube .
3.Urine if turbid is filtered and acidified and then the urine (24 hr collection ) is filled
upto the mark U and Esbachs reagent is added upto the level R.
4.Cover it with a stopper and let it stand for 24 hrs. Then the reading is taken in gms /L.
Esbachs reagent : 5 gms Citric acid
10 gms Picric acid
100 ml Distilled water .
It is a yellow coloured solution .

54
 2.Glucose:
Glycosuria along with hyperglycemia:
1. Diabetes mellitus
2.Chronic pancreatitis
3.Haemochromatosis
4.Tumour in pancreas
5.Cystic disease of pancreas
6.Hyperthyroidism.
7.Cushing syndrome
8.Acromegaly
9.Pheochromocytoma.

Glycosuria without hyperglycemia:

1.Renal tubular disease
2.Multiple Myeloma
3.Heavy metal poisoning.
Glycosuria can also be due to decreased renal threshold as in
pregnancy. Alimentary glycosuria is due to rapid absorption of glucose from GIT i.e after
partial gastrectomy.

Chemical test for glucose:

1.Benedicts test: Semiquantitative test.

Reagent: CuSO4, Na2CO3, Sodium citrate and distilled water.
Principle: Reducing substances in urine reduce CuSO4 which is blue in colour to
insoluble cuprous oxide which is yellow in colour. It detects all the reducing sugar in
urine including glucose, fructose, lactose etc, hence it is a non- specific test.
Method: Take 5ml of benedicts reagent in a test tube, boil it. Any change in the solution
indicates that solution is not fit for use. Cool the sample, add 8 drops of urine in it.Boil
for 2 min., then cool down and look for colour change.
No change in colour-Negative
Green colour- Trace ( less than 0.5 gm )
Green ppt - + ( 0.5 1.0 gm )
Yellow ppt - ++ (1.0 -1.5 gm )
Orange ppt -+++( 1.5 2.0 gm )
Brick red ppt- ++++ ( more than 2.0 gm )

2.Fehlings test
Advantages of Benedicts test over Fehlings test:
1.Single solution test
2.Stable solution
3.Much more sensitive
4.To differentiate other reducing sugars from glucose we do Osazone and Seliwanoffs
test.
5.Other non sugar reducing substance which give positive Benedicts test are uric acid,

55
creatinine and salicylic acid-False positive test
3.Reagent strip test:
1. These strips are coated with glucose oxidase and the test is based on enzymatic
reaction.
2. Test is specific for glucose.
3. Strip is dipped in urine.
4. If there is change in colour of strip , it indicates presence of glucose.
5. Colour change is matched with standard colour chart provided on the label of the
reagent strip bottle.

3.Ketones:
1. These are metabolic products of fat .
2. They are acetone (2%) ,acetoacetic acid(20%) and b-hydroxybutyric acid (78%).
3. Ketosis means increased ketones in blood and urine . Diabetic Ketoacidosis-Found in
uncontrolled diabetes mellitus
4. Ketonuria will occur before there is a significant increase of ketones in blood .
Non diabetic Ketonuria-Starvation ,Severe vomiting ,diarrhoea and eclampsia.
Lacticacidosis and Ketonuria-Occurs in acute renal failure ,salicylate poisoning.

Tests for ketonuria:

1.Rotheras test -Rotheras test detects acetone and acetoacetic acid.
2.Gerhardts test -Gerhardts test detects b-hydroxybutyric acid.
3.Reagent strip test

Rotheras test:
Reagents:
1.Ammonium Sulphate
2.Liquor Ammonia
3.Sodium Nitroprusside
Principle: Ketones in urine form purple colour with Sodium Nitroprusside.This test is
positive for acetone and acetoacetic acid .beta- hydroxybutric acid is not detected by it .
Method :
1.Take 5 ml of urine in test tube.
2 Saturate with ammonium sulphate ..
3. Add a crystal of sodium nitroprusside and shake it .
4.Pour liquor ammonia from side of test tube .
5 A purple ring is formed at the junction of the two layers .
Ketone is lost into the air if sample is left standing at room temperature ,so urine
should be tested immediately .

4. Blood:

Blood in urine is known as haematuria . In microhaematuria ( occult blood ) there is

such small amount of blood that there is no colour change and is detected only
microscopically or chemically .

56
.
Causes of haematuria:
1.Acute glomerulonephritis
2.Trauma to urinary bladder
3.Tumor of urinary system
4.Tuberculosis of urinary system
5.Calculus
6.Sometimes in nephrotic syndrome
7..Renal infarction

Haemoglobinuria;Hb in urine as a result of i.v. haemolysis

Causes of haemoglobinuria :
1. Prosthetic cardiac valve
2.Severe exercise
3.Extensive burns
4.Marching or running for prolonged time
5. Bacteria or parasite like malaria and C. Welchii.
6.Toxins like cobra venom.
7.Alkaline urine can also cause lysis of RBC releasing Hb
.
1.Benzidine Test:
Principle: Peroxidase activity of haemoglobin from the lysed blood cell due to glacial
acetic acid releasing reactive 02 species from the H2 02 which oxidizes benzidene to a
compound of blue green colour .
Method:
1. Take 2 ml of urine in test tube .
2. Benzidine solution: Pinch of benzidine powder in 1 ml of glacial acetic acid is
prepared. Add 1 ml of benzidine solution .
3. Add 1 ml of H202 by side of tube .
4. Appearance of dark blue green colour indicates presence of blood .
5. If urine sample is contaminated by bacteria ,then bacterial peroxidase may convert
H2O2 to [O] giving a false positive reaction .

2.Orthotoluidine test:
Method A pinch of O-toluidine is dissolved in 2 ml of glacial acetic acid in a test
tube .Add 10-15 drops of urine and shake well .Add 4 ml of 30 % H2O2.
Result A greenish blue colour indicates the presence of haemoglobin or occult blood.

3.Reagent strip test: Dip the strip, coated with orthotoluidine in the urine. If changes to
blue colour then blood is present.

5.Bile pigments :Present in urine in form of conjugated bilirubin.Conditions in which

bilirubin is present in urine are:
1. Hepatocellular jaundice
2. Obstructive jaundice.

57
Fouchets test :
Reagent : Fouchets reagent-Trichloroacetic acid & ferric chloride ,Barium chloride.
Principle:Barium chloride reacts with sulphates in urine to form barium sulphate which
are large sized particle which adsorbs bilirubin.This gives a blue green colour after the
reaction .
Method :Take 2 ml of urine in a test tube . Add 2 ml of Barium chloride. Filter it . Spread
the filter paper.Add 1-2 drop of Fouchets reagent .Appearance of blue green colour
denotes bile pigment in urine. The ferric chloride oxidizes bile pigment bilirubin to green
coloured biliverdin.

6.Bile salts:
They are taurocholates and glycocholates.
Hay sulphur powder test:
Principle: Bile salts decrease surface tension of urine .
Method: Take a small beaker /large test tube and fill it with urine.Sprinkle sulphur
powder on top .If sulphur sinks, it confirms presence of bile salts in urine .
Precaution:Tube must be completely filled with urine
The test is positive in :
1.Infective hepatitis
2.Obstructive jaundice
3.Acute hepatitis

58
Objective :To study the microscopic examination of urine
It is done by centrifuging 10-15 ml of urine at moderate speed (1500 r.p.m.-2000 r .p
.m. for 5 minutes ) .
1.Urine must be examined fresh.
2.A thin preparation of sediment after throwing supernatant under the coverslip without
any air-bubble must be taken.
3.The condenser should be low and light cut down with partial closure of diaphragm
while examining in low power.
4.Use low power , high power only when necessary to study finer details 5.Before
centrifuging,see that the urine in the container is well mixed or deposits may have settled
to the bottom leaving the supernant urine clear .

It gives two types of sediment:

Organised
Unorganised

Organised sediment:

1.Cells:
They are RBC (round discoid structures) , WBC or Pus cells (degenerated polymorphs)
which are 0-4 pus cells / hpf seen , epithelial cells , parasites, bacteria, spermatozoa.

2.Casts:
Cast formation takes place in distal and collecting tubule as it requires acidic Ph and high
solute conc. They dissolve in alkaline medium .Casts have parallel sides and blunt
ends.They are formed by coagulation of albuminous material.(Tamm horsfall protein)
For seeing casts in microscope , condenser has to be brought down , better seen in
phase contrast microscope .
Types of casts:
Hyaline cast: Homogenous, made of coagulated protein material .It looks semi-
transparent and colourless . Increased number is seen with renal disease and transiently
increased with exercise , fever,CHF and diuretic therapy,chronic renal disease and
malignant hypertension.
Granular cast: Granular with remnants of different types of cells which have
disintegrated or degenerated . Indicates kidney disease .Not found in normal urine.
Red blood cell cast: These are cast with RBCs embedded in the coagulated protein in
tubules . Found in acute nephritis and bacterial endocarditis .
Epithelial cast: These are cast with epithelial cells embedded in coagulated protein in the
tubule . Found in acute nephritis .
Pus cell cast:These are found in suppurative conditions of the kidney .They are formed
by the pus cells being embedded in the coagulated proteins ,seen in chronic
pyelonephritis.
Fatty cast:These are casts in which are embedded numerous fat globules .
Waxy cast:These are like hyaline casts , which have dull waxy appearance. They are

59
found in terminal stage of nephritis.
.
Unorganised sediment :

Crystals in acidic urine:

a.Calcium oxalate: Seen in normal urine .Envelope shaped or dumb bell shaped and seen
when calculi are present .Source of it is tomato spinach.
b.Uric acid or urate crystal: Present in normal urine if kept for long time .Seen as
yellow brown small granules .
c.Cystine crystals : Refractile,double hexagon,presenting cystinuria.
d.Leucine and tyrosine: Leucine crystals are yellow ,oil-appearing spheres.Tyrosine
crystals are fine silky needles which are arranged in sheaves or clumps .They may appear
black.They usually occur together and found in conditions associated with severe liver
damage.
e.Sulpha drugs:They are of variable shape seen after intake of S-drugs .other crystal
found is cholesterol crystal.

Crystals in acidic urine

S. No. Crystal Appearance Conditions

1. Calcium oxalate Envelope or dumb bell Calculi, ingestion of lot of oxalate

shaped containing food tomato, spinach
2. Uric Acid Yellow Brown, single or Gout
rhomboid
3. Amorphous Yellowish brown small Present in urine when kept for long
urate granules time, no clinical significance
4. Cystine crystals Refractile, colourless Congenital cystinosis
hexagonal plates
5. Leucine and Yellow, oil-appearing Liver damage
tyrosine spheres. Tyrosine crystals
are fine silky needles
arranged in sheaves
6. Cholesterol Geometrical shape Nephritis, tissue breakdown
7. Sulphonamide Rosettes or roundels Sulpha drug precipitates

Crystals found in alkaline urine:

a.Phosphates: Phosphates of ammonium and magnesium . Coffin lid shaped or flat fern
appearance . May indicate stone in kidney or bladder .
b. Biurates: Opaque yellow crystals . They are in the form of sphere with spikes on the
wall of sphere.(Thorn apple).
Other crystal found is calcium carbonate .

60
Crystals in Alkaline Urine

S. No. Crystals Appearance Conditions

1. Triple phosphate (ammonium Colourless prism with Stone in kidney or
magnesium phosphate) oblique ends. bladder, chronic
cystitis
2. Amorphous phosphate Amorphous granular Normal urine
3. Calcium carbonate Smell, colourless sphere No clinical
or dumb bell significance
4. Calcium phosphate Long, thin, colourless Stone formation/
prism with one pointed Normal urine
end
5. Biurates Yellow crystals, like Found in long
thorn apple standing urine

Cylindroid: Resemble casts but one of the end is tapered .

Telescoped sediment:
This term is used to describe simultaneous occurrence of elements of acute and chronic
glomerulonephritis as well as of nephrotic syndrome in urine.It may include RBC,RBC
cast ,cellular casts,waxy,lipid droplets,fatty casts.
Such sediments may be found in lupus nephritis and SABE.

References:
Practical clinical biochemistry by Harold Varley

61
Objective :To study and draw the various instruments used in clinical pathology and
haematology.

Urinometer
Specific gravity of urine (1.003-1.030) can be measured by urinometer which is
calibrated for a temperature correction also. For every 3 degree celsius rise in room
temperature beyond the calibrated temperature (usually 20 degree celsius ) ,0.001 is
added to the recorded reading and 0.001 is subtracted for every 3 degree celsius fall in
temperature . 15 ml urine sample or a 24 hrs urine sample can be taken .

Precaution:
Urinometer should not touch the walls of container and container shoild be filled upto the
brim.

Specific gravity is increased :

1.Dehydration
2.Restricted fluid intake.
3.Diarrhoea
4.Vomiting
5.Fever
6.Excessive sweating
7.Diabetes mellitus
8.Albuminuria
9.Acute glomerulonephritis

Specific gravity is decreased :

1.Excessive fluid intake
2.Diabetes Insipidus
Low and fixed specific gravity (1.010) is seen in chronic renal failure .

Esbachs albuminometer
Protien excretion in a 24-hr urine sample is carried out using Esbachs
albuminometer.Preservatives are added.

Method:
1. Fill the Esbachs albuminometer tube to mark U with urine.
2. Pour Esbachs reagent up to mark R.
3. Mix the two thoroughly and keep for24 hrs.
4. Measure the precipitate level in gms /litre of urine.
5. >150/24hr is clinically significant.

62
Causes of proteinuria:
Kidney diseases like-1.Nephrotic syndrome
2.Acute glomerulonephritis
3.T.B.of kidney
4.Renal cell carcinoma
5.Renal vein thrombosis
Others are :1.Muscular exertion
2.High fever
3.Heavy metal poisoning
4.Orthostatic albuminuria

Orthostatic albuminuria occurs only when the patient is active on his feet ; early morning
sample is negative for protein.

Lumbar Puncture Needle:

CSF is formed within the ventricles and circulates in the subarachnoid space and in the
ventricles.Total volume of CSF is 125-150 ml .CSF is obtained by lumbar puncture.
Needle is composed of a stylet and needle and is used for collection of CSF and other
body fluids. The needle is 10-12 cm in length and is made up of platinum or German
alloy. The stylet of the needle has a pin which fits into the slot of the head of the needle to
lock it in position.

Site: L3 - L4

Procedure :
1.Make the patient lie on right or left lateral position with legs and neck flexed with
knees and chin approximated.Back of the patient should be at the edge of the bed .Feel
for the iliac crest
2..Draw a line from the highest points of iliac crest down across the back .This crosses
the 4th lumbar spine or the space between L3-L4 lumbar spines.Mark the area between
L3-L4lumbar spines.
3. Clean the area around L2-L5 with iodine followed by alcohol twice .
4.Inject 1% lignocaine into skin over L3 and L4 taking aseptic precautions .
4. Then thrust the needle towards the centre of intravertebral space injecting the solution
as needle goes deeper .
5.Wait for 5 minutes for the area to be anaesthesised.
6..Introduce the sterile LP needle with stylet firmly through the skin in the middle line
between L3-L4 spines.When the needle enters the spinal cavity ,the resistance gives
away.
7.Withdraw the stylet.CSF starts flowing from the needle .
8.Collect CSF into sterile bottles.

Indications:
A. Diagnostic:
1. Meningitis.

63
2..Encephalitis
3.Subarachnoid haemorrhage
4..Gullain barre syndrome.
5..Spinal cord tumors.
6.For malignant cells / lymphoma cellsin suspected metastatic deposits in brain
/meninges.
7..To inject radio-opaque dye for myelography.

B.Therapeutic:
1.Spinal anaesthesia
2.Chemotherapeutic drugs are given intrathecaly for CNS prophylaxis / relapse. ALL
,lymphomas,meningitis.

Contraindications:
1.Any patient with marked increase in CSF pressure (as diagnosed by papilloedema)
2.Patient with brain tumor
3.Disseminated sclerosis
4. Any infective lesion .

Complications of Lumbar puncture:

1.Herniation of cerebellum through foramen magnum.
2.Extradural / subdural haematoma.
3.Introduction of infection by passing the needle through infected skin ,subcutaneous
tissue.

Bone marrow aspiration needle:

Sites for bone marrow aspiration:
Sternum,posterior.superior iliac spine,iliac crest,anterior superior iliac spine and spinous
process of lumbar vertebra.Upper end of tibia is the ideal site for marrow aspirate in
infants (tibial tuberosity) ,calcaneum.

Needles:
1. Salah needle
2. Klima needle
3. Islams bone marrow aspiration needle (T bar handle )
Bone marrow aspiration needle is made up of hard stainless steel about 7-8 cm long
with a well fitted stylet, an adjustable guard.For reusable needles, the point of the needle
and edge of the bevel must be kept well sharpened.

Method:
1.Patient is made to lie in lateral position and the legs are flexed and thighs taken against
abdomen so that posterior superior iliac becomes prominent.
2. Skin covering posterior superior iliac spine and surrounding area is cleaned with iodine
,followed by alcohol and the area is draped,taking aseptic precautions.
3. 5 ml of xylocaine is injected into the skin overlying the posterior superior iliac spine
and also the periosteum.

64
4. A small 3 mm cut is given with a sterile blade on the skin .
5.The gaurd on the needle is adjusted taking into account the thickness of the
subcutaneous tissue.
6.Salah needle along with its stylet and guard is introduced through the skin ,cut into the
bone with a rotatary clockwise and anticlockwise movement.
7.The needle is pushed through the cortex into the medullary bone and resistance gives
away as needle enters the medullary cavity. The guard prevents further pushing in of the
needle .
8.The stylet is withdrawn and a 10 ml syringe is attached to the needle: .suction is applied
to draw 0.2 to 0.4 ml of marrow into the syringe .
9.The needle and syringe together are withdrawn and marrow is poured onto the slides
placed at anangle of 30 degree so that blood present in marrow is drained off. 10.Make
thin smears of the marrow.

Indications of bone marrow aspiration:

1.Red cell disorders-Megaloblastic anaemia,Pure red cell aplasia,Pancytopenia.
2.White cell disorders-subleukemic/aleukemic leukemia, all acute blastic leukemias
for( FAB typing).
3.Megakaryocytic disorders-ITP and other thrombocytopenias.
4.Myeloproliferative disorders-Polycythemia vera,CML,idiopathic thrombocytopenia.
5.Myelodysplastic syndromes.
6.Storage disorders-Gauchers and Niemann picks disease.
7.Parasitic disorders Kala-azar.
8.Plasma cell disorders Multiple Myeloma
9.For evaluation of iron stores.
10. For metastatic deposits-Ca breast ,Prostate,lung,kidney etc.
Dry tap-In Aplastic anaemia and Myelofibrosis,marrow is not obtained and needle and
syringe are dry tap .In Aplastic anaemia marrow has been replaced by fat while in
Myelofibrosis ,it is replaced by fibrous tissue.

Westergrens Pipette:

1. It is a 30 cm long pipette with diameter of bore being 2.5 mm.

2. The lower 20 cm is graduated from 0-200 from top to bottom.
3. It is used in westergrens method of ESR estimation.
4. Anticoagulant used is 3.8% trisodium citrate in a ratio of 1:4 i.e.1 ml of anticoagulant
is used for 4 ml of blood.
5. We place the pipette filled with anticoagulated blood upto 0 mark in the stand
vertically and measure height of the plasma column after 1 hr.

Normal range: Men 0-15mm / 1st hr

Women-0-20mm /1st hr.

Advantages:
This is a more sensitive and accurate method as compared to wintrobes method
because the column is longer i.e. 200 mm.

65
Precautions:
1. The tube should not be inclined, should be vertical .
2. Ratio of anticoagulant to blood should be 1:4.
3. There should not be any clot in the blood sample .
4. No air bubbles, no vibrations.
5. Check for haemolysis.

Wintrobes Tube:

1.This tube is used for estimation of ESR first as well as centrifuged for estimation of
haematocrit/PCV
2.EDTA blood is used .
3.First the tube is filled with the help of Pasteur pipette upto the mark 0.
4.Then it is kept vertically and reading is taken after 1 hr .

Normal range: 0-7 mm in 1st hr male

0-14mm in 1st hr female.

Advantages:
1. Wintrobes method is a simple method and small amount of blood is required.
2. After measuring ESR ,the same tube can be centrifuged to determine PCV.
3. Smears of buffy coat can be made in cases of leucopenia,subleukemic / aleukemic
leukemia and for LE cell test.

Sources of error:
1. Error due to introduction of air bubble .
2. Error due to hemolysed sample.
3. Heparin alters RBC membrane potential and should never be used.

Haemoglobinometer: (Sahlis method)

It consists of :
1.Hemoglobinometer pipette with a mark of 20 ul.
2.Hemoglobinometer tube graduated in % at one side and gms/100 ml on the other side.
3.A comparator with a pair of standard non-fading brown color glass with a space in
between for the hemoglobinometer tube .
4.N/10 HCL.
5.Dropper
6.Glass rod used as a stirrer.

Principle:
Blood is added to N/10 HCL which converts hemoglobin into acid hematin .Brown
colour of acid hematin is matched against the brown colour of the comparator .

66
Advantages:
1. It can be used as a bed side and O.P.D.
2. Cheap and easy
3. No extra instrument is required.
4. No power supply is needed.

Disadvantages of sahlis method :

1 .Since it is a colour matching technique , there may be visual error in matching .
2 .After 10 min , colour of acid hematin starts fading , therefore the Hb value is slightly
lower than the actual Hb of individual .
3. Acid does not convert all haemoglobins into acid hematin ,therefore haemoglobin
value obtained is slightly lower than the actual hemoglobin of the individual.
4. Brown colour of comparator also fades after sometime.

Normal values:
Men-13-16 gm%
Women-12-15 gm%
Newborn-13.5-19.5 gm%
Children(upto 6yrs)-11-14 gm%

Neubauers Chamber:

Most commonly used counting chamber is levy chamber with improved neubauer .
Identification:Chamber has 2 ruled stages separated by a small gutter .The 2 stages in
turn are separated from 2 ridges by gutter ,one on each side.
Ruled area: It is of 3x3mm on each chamber stage which is divided into 9 squares.Big
spaces at 4 corners measure 1x1 mm each and each square is divided into 16 small
squares .These are used for counting WBCs
Central 1x1 mm square is divided into 25 squares ,each of which is in turn divided
into 16 small squares. Area of each 25 small squares measures 1/5x1/5 mm.. Five of these
medium squares i.e. 4 corner squares and one central square is used for RBC count
.Depth of grooved area is 1/10 mm so that the total volume of each big square is 1x1x0.1
mm=0.1 cumm. Volume of medium square in central area is -1/5x1/5x1/10=1/250 cumm

Calculation:
TLC/cumm-No of WBC counted x Dilution
Area counted x Depth
N= No. of cells counted in all four outer squares .
Dilution = 20
Area counted= 4 x 1 x 1 sq.mm.
Depth= 0.1mm
TLC= 4 x 0.1= N x 50 cells/cumm.
Calculation of RBC count = N x 200 = N x 10,000 cells/cumm.
5x1/5x1/5x1/10

67
 Other uses of neubauers chamber :
1. TLC
2. TLC of body fluids including CSF
3. Sperm count , Platelets (in low counts )
4. RBC( in low counts).

Precaution:
Major sources of error are :
1.Improper volume measurement
2.Improper charging of chamber
3.Use of defective pipette and wrong calculation .
4.Once the chamber is filled , counting should be done as early as possible before fluid
dries up and air bubbles enter the chamber .
5.Use clean and dry glassware.

Franzens aspiration syringe holder:

This is used for F.N.A.C. of superficial and deep organs.

L-moulds: They are used for making blocks. These are made up of aluminium or wood.

Cassette: They are used for processing of tissues. These are box like structures with
many holes so that chemicals can pass easily.

Block holder: During microtomy, blocks are fixed on block -holders for cutting of
sections.

Refrences:
1.Todd Sanford
2.Wintrobes clinical Haematology
3.Dacie

68
Objective :To study the H&E stained section of Acute appendicitis.

Clinical History : 10 year old child presented with pain in right iliac fossa ,fever &
vomiting .His peripheral blood smear shows polymorphonuclear leukocytosis. Clinically
rebound tenderness at MacBurnys point may be present.

General features:
1.Appendicitis is the most common cause of acute abdomen.
2.Acute appendicitis develops as a result of obstruction. Causes of obstruction are
fecolith, foreign body , calculus, gallstone ,tumor of caecum & appendix. Non-
obstructive appendicitis is secondary to generalized infection usually of viral etiology.

Gross :
1.The appendix is swollen .
2.Serosa is hyperaemic and coated with fibrinopurulent exudate.
3.The mucosa is ulcerated and sloughed.

Microsopic features :
In all stages, the most important diagnostic histological criteria is the neutrophilic
infiltration of the muscularis propria.
Early: Besides neutrophilic infiltration,congestion & edema of the appendiceal wall
occurs.
Late: 1. Mucosa is sloughed off and wall becomes necrotic.
2. Blood vessels may get thrombosed
3. There may be neutrophilic abcesses in the wall.
4. Peri appendiceal inflammation is seen in advanced cases.

69
Objective :To study the H&E stained section of Chronic cholecystitis.

Clinical Features: A 48 yrs old female patient complains of recurrent attacks of right
upper quadrant colicky pain accompanied by nausea ,vomiting & intolerance to fatty
foods.

Gross :
1.Gall bladder is thickened .
2.There is focal loss of velvety appearance of mucosa .
3.Serosa appears dull and opaque .
4. Associated with gall stones.

Microscopic features :
1.The normal mucosal folds are either diminished in size or become flattened.
2.Mononuclear infiltrates (lymphocytes and plasma cells) are seen in all the layers
including lamina propria , muscle layer and subserosal layer.
3.Muscle coat and serosa shows fibrosis.
4.At places mucosal folds outpouched into the muscle layer to form Rokitansky Aschoff
Sinuses.

70
Objective :To study the H&E stained section of Chronic appendicitis.

Clinical History : -A young boy presented with recurrent pain in right iliac fossa.The
pain is irregular in frequency, associated with nausea and low grade fever.
On examination-Rebound tenderness at macburneys point can be elicited.

Gross: A small tubular appendix which is firm in consistency .Outer surface is pale and
smooth.There is no sign of inflammation.

Microscopic Features :
1.Mucosal lining is intact with well formed mucosal glands.
2.Submucosa shows hypertrophied lymphoid follicles.
3.There is presence of transmural infiltration by lymphocytes,plasma cells and occasional
eosinophil.
4.Fibrosis is seen in subserosal & muscular layers.

71
Objective :To study the H&E stained section of T.B. lymph node.

Necrosis:
Definition: It refers to a sequence of morphologic changes that follow cell death in
living tissue .It is the result of two essentially concurrent processes
A) Enzymatic digestion of the cell
B) Denaturation of proteins

Types of necrosis:
1.Coagulative necrosis: Ischaemic necrosis (except in brain) e.g. Myocardial infarction.
2.Caseous necrosis: e.g. Tuberculosis
3.Liquefactive necrosis: e.g. C.N.S. infarction , Abscess (Pus formation)
4. Fat necrosis: A) Enzymatic-Acute Pancreatitis
B) Traumatic-Breast
5. Fibrinoid necrosis: Auto-immune diseases, arterioles in hypertension.
6. Gangrenous necrosis: Necrosis with superadded putrefaction.

Clinical History : Patient presented with low grade evening rise of temperature, reduced
appetite , loss of weight and lymphadenopathy.

Genral Features :
1. Causative organism is Mycobacterium tuberculosis .
2. It is detected by (A) Special stains for acid fast bacilli i.e; Ziehl-Neelsen stain ,
Fluorescent dye (Auramine O Rhodamine) and
(B) Grown in culture media -Lowenstein Jensen media for 6-8 weeks.
3. In immuno-suppressed patients like patients with AIDS ,T.B. is caused by atypical
mycobacterium like Mycobacterium avium intracellulare, Mycobacterium kansasi etc.

Gross: Enlarged single /matted lymph node, cut surface is lobulated with multiple
irregular whitish necrotic and calcified areas.

Microscopic features :
Section shows:
1. Many well defined granulomas consisting of epithelioid histiocytes, Langhans giant
cells, lymphocytes and fibrous tissue .
2. Caseous necrosis: It is a distinctive form of necrosis seen most often in a foci of
tuberculous inflammation .The term caseous is derived from the cheesy white gross
appearance of the central necrotic area.

Reference:
Robbins pathologic basis of diseases, 7th edition.

72
Objective :To study the H&E stained section of CVC spleen.

Clinical History : Patient presents with complaints of dyspnoea, orthopnoea &

paroxysmal nocturnal dyspnoea.

Gross:
1.Spleen is enlarged measuring 20 x15 x12.5 cm, congested, tense.
2.Cut surface is brownish red in colour.Brown spots about 3-4 mm are seen representing
fibrohemorrhagic areas called tobacco nodules.
3.Capsule may get thickened & fibrous.

Microscopic Features:
1..Splenic capsule and trabeculae are thickened .
2.Red pulp shows congestion and marked sinusoidal dilatation with areas of recent and
old haemorrhages.
3.These haemorrhages may get organized to form Gamna Gandy bodies or siderofibrotic
nodules which are deposits of haemosiderin pigment & calcium salts on fibrous
connective tissue.
4.Recent haemorrhage is evident around the nodule.

Causes of CVC spleen:

1.Cirrhosis
2. Right heart failure
3.Thrombosis of hepatic vein (Budd Chiari syndrome)
4.Thrombosis of splenic vein
5.Stenosis of portal vein

73
Objective :To study the H&E stained section of CVC lung.

Introduction: Chronic venous congestion of lung occurs in left heart failure,especially

in rheumatic heart disease( mitral stenosis ) which leads to consequent rise in pulmonary
venous pressure.

Gross:
1.The lungs are heavy, wet and firm in consistency.
2.Sectioned surface is dark-brown in colour referred to as brown induration of lungs.
3.The brown induration of cut surface of lung is due to pigmentation and fibrosis.

Microscopic features :
1.The alveolar septa are widened due to presence of interstitial oedema as well as due to
dilated and congested capillaries.
2.Septa are mildly thickened due to slight increase in fibrous connective tissue .
3.Rupture of dilated and congested capillaries may result in minute intra-alveolar
haemorrhages
4.The breakdown of erythrocytes liberate hemosiderin pigment which is taken up by
alveolar macrophages,so called heart failure cells present in the alveolar lumina(diagnosis
of CVC lung) .

74
Objective :To study the H&E stained section of various pigments.

Introduction : Pigments are coloured substances that are either :

a.Exogenous pigment :
Carbon:
Most common exogenous pigment is carbon.Aggregates of the pigment grossly
blacken the draining lymph nodes and pulmonary parenchyma ( Anthracosis) . Heavy
accumulation may induce emphysema or a fibroblastic reaction that can result in a
serious lung disease called coal workers pneumoconiosis.

b.Endogenous pigments:
It includes:
1.Melanin
2.Hemosiderin
3. Lipofuscin

1.Melanin:
It is formed by melanocytes exclusively. It is brown black pigment formed when
enzyme tyrosinase catalyses the oxidation of tyrosine to dihydroxyphenylalanine.It acts
as a screen against harmful U-V radiation. Adjacent keratinocytes in the skin (eg in
freckles) or dermal macrophages can accumulate the pigment.

2 .Hemosiderin :
It is Hb derived granular pigment ,golden yellow to brown in colour which accumulates
in tissue where there is local excess of iron. Iron is stored in association with a protein
,apoferritin ,to form ferritin micelles. Hemosiderin pigment represents large aggregates of
these micelles. It is demonstrated by Prussian blue histochemical reaction as blue black
appearance.
It is found in the mononuclear phagocytes of the liver ,bone marrow ,spleen and
lymphnodes and in the scattered macrophages throughout other organs.

3. Lipofuscin :
Lipofuscin is an insoluble brownish yellow, intracellular pigment, also known as
lipochrome and wear and tear or aging pigment. Lipofuscin is composed of polymers
of lipids and phospholipids complexed with protein suggesting that it is derived through
lipid peroxidation of polyunsaturated lipids of subcellular membranes Lipofuscin is not
injurious to the cell or its functions. Its importance lies in its being the telltale sign of free
radical injury and lipid peroxidation.

75
Objective: To study the H&E stained section of Monckebergs Sclerosis.

Introduction: Monckebergs calcification( Medial calcific sclerosis) is characterized by

calcific deposits in the degenerated media of large and medium sized muscular arteries in
elderly patients .
Sites of calcification are the extremities,genital tract, kidneys,gastric mucosa, lungs
,systemic arteries and pulmonary veins.

Gross : It produces pipe stem like rigidity of affected artery with out significant luminal
narrowing.

Microscopic features :
1.The calcium deposits have a basophilic granular amorphous clumped appearance in the
medial arterial wall .
2.Smooth muscle of media is replaced by acellular hyalinised fibrous tissue.

76
Objective :To study the H&E stained section of lipoma.

Clinical Features : Patient presents with a soft palpable mass which is mobile and non-
tender.

General Features :
1. Lipoma is the commonest tumor of adipose tissue.
2. Common sites are: neck ,back, shoulder and trunk.
3.Usually they occur singly but may be multiple.

Gross-
1. It is a small, encapsulated , rounded, multilobulated mass, soft yellow in colour
2. Deeply situated lesions are less demarcated.
3. On cutting, it is a yellowish mass with soft consistency.

Microscopic features :
1. A thin fibrous capsule surrounds the periphery.
2. It is composed of lobules of mature adipose cells separated by thin fibrous septa.
3. Cells are polygonal, vacuolated with a thin rim of cytoplasm
4. Nucleus is placed eccentrically.
5. Lesion is indistinguishable from normal fat .

Fat is demonstrated by special stains such as Sudan black, Sudan III , Sudan IV and
Oil red O on frozen and cryostat sections.
Its malignant counterpart is liposarcoma.

77
Objective :To study the H&E stained section of Fibroadenoma.

Clinical history: Patient presents with a palpable mass in one or both the breasts . the
lump is firm,freely mobile and non-tender.

General features:
1.Fibroadenoma is a common benign tumor of fibrous and epithelial elements of breast.
2.Though it can occur at any age during reproductive life, most patients are between 15
-30 years of age.

Gross :
1.Typical fibroadenoma is a small ,solitary ,well encapsulated,spherical or discoid mass.
2. The cut surface is firm ,gray white,slightly myxoid and may show slit like spaces
formed by compressed ducts.

Microscopic features :
Fibroadenoma comprises mostly of fibrous tissue.
There are two patterns:
Intracanalicular pattern-
The stroma compresses the ducts so that they are reduced to slit like clefts lined by ductal
epithelium or may appear as cords of epithelial elements.
Pericanalicular pattern
Dense stroma is seen around the intact glands which are not compressed ,hence ducts
remain patent or dilated.

78
Objective :To study the H&E stained section of leiomyoma.

Clinical History : 35 Year old female presented with excessive bleeding per vaginum
and pain in abdomen since last one year.

General Features:
1. Leiomyomas are the most common benign smooth muscle tumors.
2. Sites: Often arise in uterus where they represent the most common neoplasm in
women. They can occur within Myometrium (intramural), just beneath the
endometrium(submucosal) or beneath the serosa(subserosal). May also arise in
erector pili muscles found in skin, nipples, scrotum and labia(genital leiomyomas)
and less frequently in deep soft tissues.
3. Symptoms: Most important symptoms are produced by submucosal
leiomyomas(abnormal bleeding),compression of bladder(urinary frequency),sudden
pain if disruption of blood supply occurs,and impaired fertility.
4. These tumors are estrogen responsive and may undergo rapid increase in size during
pregnancy. Myomas in pregnancy increase the frequency of spontaneous abortion ,
foetal malpresentation, uterine inertia and post partum haemorrhage.
5. Malignant transformation (leiomyosarcoma) within a leiomyoma is extremely rare.
The most important criteria for malignancy is mitotic index.
6. Leiomyoma may undergo following secondary changes-
a.Hyaline degeneration
b.Red degeneration in pregnancy
c.Calcification
d.Fatty change
e. Infarction
f.Malignancy (rarely)

Gross:
1. Panhysterectomy specimen with distorted uterus due to multiple leiomyomas present
at various sites.
2. They are firm ,circumscribed and nodular.
3. Cut surface :gray-white and shows characteristic whorled appearance.
4. Endometrial cavity can be seen.
5. Cervix and bilateral adnexa appear to be normal grossly.

Microscopic Features :
1. Sections from leiomyoma show whorled interlacing bundles of smooth muscle cells
resembling the architecture of the uninvolved myometrium .
2. Smooth muscle cells are uniform in size and shape and have characteristic central
oval nuclei and long,slender bipolar cytoplasmic processes.
3. Mitotic figures are scarce.

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Objective :To study the H&E stained section of Benign Hyperplasia of prostate

Clinical History : 56 years old male presented with retention of urine with past h/o
difficulty in micturition .On per rectal examination ,prostate was enlarged and nodular.

Genral Features :
1. Extremely common disorder over age 50years
2. Prostate gland is markedly enlarged and nodular .
3. Characterized by hyperplasia of prostatic stromal and epithelial cells
4. Formation of large, fairly discrete nodules in periurethral region of the prostate.
5. When nodules are sufficiently large, the nodules compress and narrow the urethral
canal to cause partial, or sometimes virtually complete obstruction of urethra.

Gross:
1. Lobes of prostate glands are enlarged
2. Outer surface: nodular, smooth and firm .
3. Cut surface: grayish white with multiple nodular areas and occasional cystic spaces.
4. Appearance on cut section varies depending upon whether hyperplasia is
predominantly of glandular or fibromuscular tissue.

Microscopic features :
1. Sections of prostate show fibromuscular and glandular hyperplasia
2. Few glands show papillary infoldings
3. Glands are lined by double layered epithelium which is cuboidal to low columnar
Some acini contain corpora amylacea.
4. Corpora amylacea: eosinophilic, proteinaceous, inspissated material seen in the lumen
of the glands.

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Objective :To study the H&E stained section of Osteoclastoma

Clinical History : 38 years old male presenting with bony hard swelling of knee joint.

General features:
1. Also called as Giant Cell Tumour because it contains a profusion of multinucleated
osteoclast type giant cells.
2. Benign but locally aggressive neoplasm
3. Usually affects persons of age 20-40 years.
4. In adults it involves the epiphysis and metaphyses,but in adolescents they are
confined proximally by the growth plate and limited to the metaphysis.
5. Most are solitary,however multiple or multicentric tumors do occur especially in
distal extremities
6. Radiologically: large, purely lytic ,eccentric epiphysis(soap bubble appearance)
erode into the subchondral bone plateoverlying cortex is frequently destroyed
producing a bulging soft tissue delinated by a thin shell of reactive bone.
7. Common sites: Around the knee (distal femur and proximal tibia).But virtually any
bone may be involved. Location of these tumours in the ends of bone near joints
frequently causes patients to complain of arthritic symptoms.
8. It usually does not metastatise but local recurrence is common.
9. Giant cells are considered non -neoplastic.
10. Grade of tumor is according to mitotic index and cytological atypia of stromal
cells which is neoplastic component (Grade I-III).

Gross:
1. Tumors are large and red brown, haemorrhagic.
2. Frequently undergo cystic degeneration
3. Cut surface is gray tan with brownish and cystic areas.
4. Tumor is extending into surrounding soft tissue also.

Microscopic features :
1. Sections show large number of multinucleated osteoclast like giant cells which are
regularly scattered throughout the spindly stromal mononuclear cells .
2. Stromal cells are uniform without pleomorphism.
3. Areas of haemorrhage and necrosis are also seen.

81
Objective :To study the H&E stained section of Colloid Goitre

Clinical History : 32 years old female presents with a midline swelling in the neck since
few years . Swelling moves with swallowing/deglutition

Genral Features :
1. Goitre presents as a midline mass in the neck region
2. The dominant clinical features of goitre are those caused by mass effects of enlarged
gland
3. Goitre may cause airway obstruction, dysphagia and compression of large vessels in
the neck and upper thorax.

Gross:
1. Multinodular thyroid mass, cystic to firm in consistency .
2. Cut surface: irregular nodules containing variable amounts of brown ,gelatinous
colloid are present.
3. Partial or incomplete encapsulation of nodules.
4. Areas of haemorrhage and calcification.seen.

Microscopic features :
1. Colloid rich follicles of varying size and shape
2. Follicles lined by cuboidal to low columnar epithelium.
3. The follicles are separated by bands of fibrous tissue.

82
Objective :To study the H&E stained section of Transitional cell carcinoma.

Clinical History : A 55 years old male complained of painless haematuria and pain in
suprapubic region radiating towards the prepuce .Cystoscopy revealed a growth in the
bladder.

Genral Features :
1. Represent about 90% of bladder tumors
2. There are two distinct precursor lesions to invasive urothelial carcinoma:
3. Noninvasive papillary tumors(more common)
4. Flat urothelial carcinomas(carcinoma in situ)
5. Cigarette smoking increases the risk threefold to sevenfold.
6. Characteristically produce painless haematuria

Gross:
1. Specimen shows part of urinary bladder wall with exophytic,papillary excrescences
presenting tumor mass.
2. Papillary lesions appear as red, elevated excrescences.
3. Areas of haemorrhage present.
4. On opening the tumor was seen practically filling the whole of the cavity

Microscopic features :
1. Sections show papillary exophytic lesion consisting of long broad papillae
2. Papilla: have well-defined delicate fibrovascular core covered with transitional
epithelium which at most places is more than 7 layers thick.
3. Epithelium cells show uniform vesicular nuclei having fine granular chromatin.
4. Cytoplasm is clear to amphophillic and is moderate in amount. There is no
pleomorphism.

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Objective :To study the H&E stained section of Renal cell carcinoma.

Clinical History : 70 year old male presented with gross haematuria and frank pain.

Genral Features :
1. Most common in 6th to 7th decades
2. Men are affected about twice as commonly as women
3. These tumors are derived from the renal tubular epithelium and hence they are
located predominantly in the cortex .
4. The risk of developing these tumors is higher in smokers and those who have
occupational exposure to cadmium .
5. Also called as Hypernephroma:Because of their gross yellow colour & resemblance
of the tumor cells to clear cells of adrenal cortex
6. The three classic diagnostic features are costovertebral pain, palpable mass and
hematuria (hematuria is usually intermittent and microscopic)

Gross:
1. Tumor may arise in any portion of the kidney, but more commonly it affects the pole,
particularly the upper pole .
2. Generally large,yellow & circumscribed .
3. Cut surface: yellow to white ,with prominent areas of cystic changes, necrosis &
haemorrhage
4. The margins of tumor are well defined .
5. It may invade the renal vein and grow as a solid column within the vessel as far as
Inferior vena cava. and into the right side of the heart .

Microscopic features :
1. Depending upon the amount of lipid and glycogen present ,tumor cells of clear cell
type of renal cell carcinoma appear almost vacuolated.
2. The clear cells are demarcated only by their cell membranes .
3. The nuclei are usually small and round and pushed basally.
4. The tumor cells are arranged in solid nests separated by delicate fibrous stroma
containing blood vessels.

84
Objective :To study the H&E stained section of Squamous cell carcinoma

General Features :
1. Malignant tumour arising from areas which are lined by stratified squamous
epithelium
2. Usually occurs in elderly persons.
3. Predisposing factors are-
Skin irradiation.
Chronic ulcers over varicose veins or Marjolins ulcer
Repeated irritation of skin by chemicals like tar and dye.
Premalignant conditions like Bowens disease,Leukoplakia and Pagets disease.

Gross:
Tumour presents as ulcerated areas with elevated ,indurated rolled margins/sessile mass
polypoidal mass or fungating growth .

Microscopic features :
Invasive carcinoma of surface epidermis has following features-
1. There is irregular downward proliferation of epidermal cells into the dermis .
2. Depending upon the grade of malignancy ,the mass of epidermal cells show
pleomorphism ,nuclear hyperchromatism and abnormal mitotic figures.
3. Well differentiated squamous cell carcinoma have whorl-like arrangement of
malignant cells forming horn pearl (keratin pearl). Centre of this pearl contains
laminated keratin material.

85
Objective :To study the H&E stained section of Adenocarcinoma duodenum

Clinical History : 54 Years male presented with intestinal obstruction.

Genral Features :
1. Adenocarcinomas are malignant tumors ,arising from the secreting epithelia as well
as from the acinar lining cells of the glands.
2. Age group: 40-70 years.
3. Tumors grow in napkin-ring encircling pattern or as polypoid exophytic masses.
4. Tumors in duodenum,particularly those involving ampulla of vater may cause
obstructive jaundice early in their course.
5. Common sites of origin include stomach ,intestine ,kidneys,uterus,pancreas,gall
bladder and ovary etc.
6. Increased risk factors for adenocarcinoma small intestine:
7. Chronic inflammation associated with Crohns disease, Familial adenomatous
polyposis(FAP),Hereditary nonpolyposis colorectal cancer syndrome(HNPCC),
Peutz-Jeghers syndrome.

Gross: Part of small intestine ,long having a thickened area causing narrowing of
intestinal lumen.

Microscopic features :
1. Sections from duodenum show malignant tissue containing malignant glands of
various size and shape
2. Cells of glands show hyperchromatism and pleomorphism.
3. Malignant cells are infiltrating all the layers of duodenum.
4. Mucin secretion is minimal.
5. Intervening stroma shows mononuclear inflammatory infiltrate , areas of necrosis and
haemorrhage.

86
Objective :To study the H&E stained section of Pleomorphic Adenoma.

Clinical History : Patient presents with swelling usually at the angle of jaw, freely
mobile & non-tender .

Genral Features :
1.Pleomorphic adenoma is also called mixed tumour.
2.They represent 60% of tumours in parotid and are less common in submandibular
glands and rare in minor salivary glands .
3.They are benign in nature.

Gross : Encapsulated mass with smooth surface. On cutting, lobulated appearance with
intervening areas of grey-white gelatinous material and blue translucent areas of
chondroid seen.

Microscopic features :
1. Section shows tumor tissue with heterogenous (epithelial & mesenchymal)
appearance.
2. Tumor cells are forming ducts ,acini ,tubules and sheets of cells.
3. Epithelial cells are small and range from cuboidal to spindle shape.
4. They intermingle with loose myxoid connective tissue and in few sections, islands of
chondroid may be seen.

87
Objective :To study the H&E stained section of Dermoid cyst / Mature teratoma.

Genral Features :
1. Teratomas tumours are of germ cell origin.
2. Teratomas are divided into 3 categories
Mature (benign)
Immature(malignant)
Monodermal or highly speacialised.
3. They arise in the 1st two decades of life and the younger the patient ,the greater is
likelihood of malignancy.
4. Most benign teratomas are cystic.
5. These neoplasms are invariably benign and are presumably show differentiation to all
the three types of germ lines. Particularly ovarian teratoma show diff. principally
along the ectodermal lines (sebaceous gland, tooth, hair follicle). To create a cystic
tumour.
6. These tumors are prone to undergo torsion (10-15% cases) producing an acute
surgical emergency

Gross:
1. An oval, multiloculated cystic mass which on cutting shows pultaceous material
(keratin, sebum, hair).
2 Cyst wall is thin and translucent.
3 At places chalky material is stucked to cyst wall.
4 Tuft of hair is & tooth may be found. Some times, tooth may be located in nipple like
protruberance, covered by hair, called Rokitansky Protruberance

Microscopic Features:
1.The cyst wall is lined by stratified squamous epithelial cells .
2.Numerous hair follicles ,sebaceous glands and sweat glands (ectodermal) are seen. At
places cartilage is present.
3.There is no evidence of malignant transformation.

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Objective :To study the given H&E section of seminoma.

Genral Features :
1. It is the commonest malignant testicular tumor.
2. Seminoma commonly occurs in 3rd and 4th decade of life.
3. It is the most important cause of firm ,painless enlargement of testis .
4. It is not seen in children .
5. It may be unilateral or bilateral.
6. It corresponds to dysgerminoma in the female ovary.
7. They are of 3 types:
a.Classic
b.Spermatocytic
c.Anaplastic

Gross :
1. Testis is enlarged symmetrically upto 2-3 times its normal size.
2. On cutting ,seminomas are large, soft,well-demarcated.
3. They appear as either a circumscribed mass or replace the entire testis.
4. It is usually homogeneous ,gray-white ,lobulated & bulges from the cut surface of the
affected testis.
5. Necrosis & haemorrhage are rare.The presence of haemorrhage indicates an
associated non-seminomatous germ cell component.

Microscopic features :
1.Tumour cells are often arranged in small lobules with intervening fibrous septa .
2. Lymphocytic infiltrate is usually present in the fibrous stroma.
3. Seminomas shows large cells with distinct cell borders,clear cytoplasm and large
round nuclei with 1-2 prominent nucleoli.Cytoplasm contains glycogen which stains
positively with PAS reaction.

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Objective :To study the H&E stained section of Hydatidiform mole.

Clinical History : Patient presented with amenorrhoea uterine bleeding accompanied

by passage of grape like structures.

Genral Features :
1. The hydatidiform mole is a common complication of gestation.
2. It is characterized by cystic swelling of Chorionic villi accompanied by trophoblastic
proliferation.
3. Once the diagnosis is made, the mole must be removed by thorough curettage After
curettage B-HCG estimation is essential to ascertain complete removal of the mole A
follow up of the patient is also necessary because app.2% complete moles may show
malignant transformation i.e. choriocarcinoma.

Gross : Specimen consists of thin walled translucent cystic grape like structures. No
normal foetal parts are seen.

Microscopic:
1.The Chorionic epithelium shows striking proliferation of both cytotrophoblast and
syncytial trophoblast.
2. There is hydropic swelling of Chorionic villi.
3. The central substance of villi is avascular,loose,myxomatous,oedematous stroma.

90
Objective :To study the H&E stained section of Rhinosporidiosis

Genral Features :
1. Rhinosporidiosis is caused by a fungus, Rhinosporidium seeberi.
2. Typically, it occurs in a nasal polyp but may be found in other locations like
nasopharynx, larynx and conjunctiva.
3. The disease is common in India and Sri Lanka.

Microscopic features
1. Section of nasal polyp is loose oedematous tissue covered with pseudostratified
ciliated columnar epithelium (respiratory type of epithelium )which at places is
ulcerated.
2. Sub-epithelial tissues contain numerous sporangia of varying size containing many
fungal spores of about the size of erythrocytes.
3. Occasionally, sporangium is broken and released spores are seen in surrounding loose
tissue. alongwith varying number of inflammatory cells like lymphocytes ,plasma
cells and eosinophils.

91
Objective :To study the H&E stained section of Actinomycosis

General Features :
1. It is a chronic suppurative disease caused by anaerobic bacteria, Actinomycetes
israelli.
2. The organisms are commensals in the oral cavity,alimentary tract and vagina.
3. The infection is always endogenous in origin and not person to person.
4. Depending upon the anatomic location of lesions,actinomycosis is of 4 types-
a. Cervicofacial actinomycosis-commonest form (60%)
b. Thoracic actinomycosis
c. Abdominal actinomycosis
d. Pelvic actinomycosis

Microscopic features -
1. The inflammatory reaction is in a form of granuloma with central suppuration. There
is formation of an abcess in the centre of lesions and at the periphery are seen
chronic inflammatory cells.
2. The centre of each abcess contains the bacterial colony consisting of a central zone of
radiating filaments and periphery of palisading,eosinophilic club shaped ends
representative of secreted immunoglobins. This is surrounded by a layer of
neutrophils,lymphocytes and plasma cells.
3. Bacterial stains reveal the organisms as gram +ve filaments,non acid-fast which stain
positively with Gomoris methenamine silver (GMS) staining.

92
Objective :To study the H&E stained section of Cysticercosis

Genral Features :
1. Cysticercosis is infection by the larval stage of Taenia solium (pork tape worm).
2. The adult tape worm resides in the human intestine.
3. Man is the definitive host and pig is the intermediate host.
4. The eggs are passed in human faeces which are ingested by pigs or they infect
vegetables .
5. These eggs then develop into larval stage in the host,spread by blood to any site in the
body and form cystic larvae termed as cysticercus cellulosae.
6. Human beings may acquire infection by larval stage by eating undercooked pork also
called measly pork or by ingesting uncooked contaminated vegetables and sometimes
by autoinfection.
7. This is not so prevalent in muslim community as pork is prohibited on religious
grounds .
8. The larvae cysticercus cellulosae presents as subcutaneous or intramuscular nodules,
though they may localize in any organ including brain causing epileptic fits .
9. Most common sites are brain, skeletal muscle and skin.

Gross:
They are seen as pearly white transluscent cyst upto 1.5 cm in diameter with an
invaginated scolex with birefringent Hooklets.

Microscopic features :
1. Section shows cross section of cysticercus larva containing fibrous pseudocapsule
which is typically infiltrated by lymphocyte, plasma cells and numerous eosinophils.
2. In some cases active granulomatous reaction is seen.

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