SAPTHAGIRI INSTITUTE OF MEDICAL SCIENCES & RESEARCH CENTER
NO.15,CHIKKASANDRA, HESARAGATTA MAIN ROAD, Bangalore-90
DEPARTMENT OF PATHOLOGY
This is to certify that this is the bonafide Pathology Practical Record of
He/ She has satisfactorily Completed the Pathology Practical as prescribed by Rajiv Gandhi
University Of Health Sciences, Bangalore during the year 201 - 201
His / Her University Registration No. is_____________________________________
Lecturer/Demonstrator Professor and Head
Department of Pathology
INSTRUCTION TO STUDENTS
1. Students should come to the practical class with buttoned apron and long hair should
either be tied up or covered by apron.
2. Students will sit in the serial order of their roll numbers and shall work in small groups
allotted under one member of the teaching staff.
3. Students should bring with them their practical record books and also either the class
notes, or the text books. Protocols for practical classes are announced in advance in each
4. Students should come prepared with theoretical knowledge of practical exercise of the day.
5. Students must possess lead-pencil and variety of coloured pencils.
6. Before starting the day’s work every student must check up the microscope given to him;
clean the eye piece and objectives and adjust the tube length, condenser, diaphragm and
mirror correctly. After the work the lenses should be cleaned, particularly the oil
immersion, and see that the lowest objectives is left in alignment. Any defect found should
immediately be reported to the teaching staff.
7. Sketches and diagrams should be neatly drawn in the same colours as seen in the
microscope filed, paying attention to relative size of cells etc.,
8. As there is little time for repetition of practical work, the two hours should be fully utilized
by repeating exercises several times and drawing as many diagrams as possible.
9. Hands contaminated with infected material should immediately be washed with antiseptic
solution and soap provided. Contamination of work tables should immediately be reported.
10. Every student should spend the major time in the given class work and the reminder can
be spent by turns on the demonstration arranged at the side table.
11. Every student should show his day’s work including, diagrams in the practical record book
and must get them signed by the teacher with date, before he can get his attendance for
the day’s practical class. It is student’s responsibility to earn the required percentage of
12. Loud talking and unnecessary movement of students from place to place should be
13. At the end of this session the record book completed in their own handwriting should be
submitted for scrutiny and certification by the Professor for having completed the course
TABLE OF CONTENTS
Sl.No. Date EXERCISE Page No.
TABLE OF CONTENTS
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TABLE OF CONTENTS
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TABLE OF CONTENTS
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PART I GENERAL WHITE
PART II HAEMATOLOGY PINK
PART III CLINICAL PATHOLOGY LIGHT YELLOW
PART IV CYTOLOGY LIGHT GREEN
PART V HISTOPATHOLOGY
GENERAL- LIGHT VIOLET
PART VI POST MORTEM LIGHT BLUE
PART VII SPOTTERS
SCHEME OF EXAMINATION
ANTONY VAN LEEUWENHOEK (1632 – 1723)
Antony Van Leeuwenhoek (Pronounced Layu-wen-hook) was born on
October 24, 1632 in Delft, Holland. Leeuwenhoek received only an elementary
education. After reading the book, Micrographia, he became interested in
microscopes and made over 500 microscopes. In 1674, he made the first
observation of microbes, becoming one of the greatest seminal discoveries in
history. He described many types of bacteria and protozoa and calculated their
sizes. In 1677, he became the first person to describe spermatozoa and was one of
the earliest to describe red blood corpuscles. In 1680, he was elected a fellow of
the Royal Society of England and was also a corresponding member of the
Academy of Sciences in Paris. Antony a Leeuwenhoek died on August 30, 1723.
ORGANISATION AND FUNCTIONING OF THE DIAGNOSTIC LABORATORY
Of better functioning of laboratory it is organized in to sub divisions
1) Histopathology; it deals with the study of tissue removed from the living body which
includes study of tissue by paraffin embedding technique and by frozen section for rapid
diagnosis. This technique includes study of structural changes observed by naked eye i.e
gross or macroscopic changes. The changes detected by light microscopy and electron
microscopy are supported by numerous special staining methods which includes
histochemical and immunological techniques, to arrive at most accurate diagnosis.
2) Cytopathology-it includes study of cells shed off from the lesion-exfoliative cytology
and fine needle aspiration cytology (FNAC) of superficial and deep seated lesions for
3) Haematology-deals with disease of the blood, work with whole blood to do full blood
counts and blood films as well as many other specialsed tests.
4) Clinical pathology-analysis of biochemical constituents of blood, urine, semen, C.S.F,
and other body fluids.
5) Immunology-Detection of abnormalities of immune system of body comprises
6) Molecular pathology-detection and diagnosis of abnormalities at the level of DNA of a
cell, is included in molecular pathology.
EXERCISE-VISIT TO CENTRAL LABORATORY
Microscope is essentially an optical instrument used for magnifying minute objects
in order to study the details of the structure.
The simplest form of a microscope consists of a single convex lens mounted onto a
ring. The lens is held at an appropriate distance from the object and thus magnification
is brought out. Usually a magnification of 10x is obtained.
The compound microscope, which is widely used in the laboratory, consists of the
eye pieces, objectives and a graduated tube which holds them in proper separation. The
instrument is focused by a rack and pinion with both the coarse and fine adjustments.
The tube is mounted on a stand with a mechanical stage to support the specimen. There
is a mechanism under the stage consisting of a condenser, an iris diaphragm and a
mirror for receiving and regulating the amount of light from a particular source.
Objectives are usually three in number, and they are designated according to their focal
lengths as under:
1. 16 mm.or2/3” - Low power
2. 4 mm. or 1/6” - High power – dry
3. 2 mm. or 1/12” - High power – oil immersion
The eye-pieces usually give 5x to 15x magnification.
Magnification of the microscope depends on. (1) The focal length of the objective.
(2)Magnifying power of the eye piece and (3) distance between the objective and the image
produced. Optical tube length is usually kept at 160 mm. Thus the total magnification
can be calculated by a formula:
Optical tube length (160 mm) X Eye piece magnification
Focal length of the objective
Oil Immersion objectives: In case of oil immersion objective, the space between
the outer lens of the objective and the object is filled with and oil like cedar wood oil
which has the same refractive index as that of glass (1.51). The rays of light do not
undergo refraction but pass into the objective giving a bright, clear image. For critical
study of bacteria, blood cells etc., where in high magnification and resolution is required,
the oil immersion lens is of much help.
Definition is the capacity of the objective to render the image distinct, eliminating
chromatic and spherical aberration.
Spherical Aberration: The rays passing through the periphery of the lens focus on
the axis at a shorter distance than those passing through the central portion of the lens
with the result that the image is distorted.
Chromatic aberration: It is caused by splitting of a white ray into its component
colour when it refracts through lens the violet-blue rays focusing nearest and red rays
farthest from the lens rendering the image fringed with colours.
Both these aberrations can be corrected by the combination of lenses of different
Resolution is the ability of an objective to distinguish minute structures. Limit of
resolution is a minimum distance between two points an objective can form separate
images of and this interval with the visible spectrum is about 0.2 µ.
Resolving Power of an objective depends upon the wave length of the light used for
illumination. The shorter the wave length the greater is the resolution. Ultra violet rays
increase the resolution and although the image is invisible, it can be photographed by
Numerical Aperture indicates the amount of light which enters an objective from a
point in the microscope field. It is defined as the ratio of the diameter of the lens to the
focal lengths and is expressed as:
NA = n sine µ
Where ‘n’ is refractive index of the medium,
2 µ= angle of aperture or angle formed by the two
extreme rays of light which starting from the
centre of the objective reach the eye. Thus ‘n’ being
constant, NA depends upon half the diameter of the lens.
The theoretical limit of 2µ is 180 in case of a dry objective where the refractive index
of air is ‘1’ and NA 1 sine 900=1.
When oil is used, n = 1.5 X sine 900 = 1.5
In general practice, 16 mm, objective should have N.A. of 0.25: 4 mm. Objective N.A.
of 0.65; and 2 mm. Objective N.A of 130.
Condenser is used for focusing light on the object. The commonly used type
condenser consists of a system of convex lenses. Lowering of the condenser diminishes
light where as raising increases.
The Iris Diaphragm: Controls the angle of light which passes into the condenser.
Mirror: The plane side of the mirror is used along with condenser and concave
METHODS OF ILLUMINATION
1. Transmitted illumination or `direct illumination: The object and its structural
details will be visible if it is coloured naturally or artificially stained.
2. Reflected illumination: When examining opaque specimens the light is thrown onto
the specimen from above. The light reflected from the specimen enters the objective.
3. Dark Ground illumination: In case of living bacteria, spirochete etc., which are
almost invisible when examined by transmitted light, dark field illumination, is used. By
means of a special type of condenser, the specimen is illuminated from below by oblique
light. Hence, only the rays that are reflected from the object enter the objective. As a
result, the organisms appear bright in a dark back ground.
CARE AND USE OF MICROSCOPE
Clean the microscope with a clean soft cloth. The objectives and the eye piece
must be cleaned with a lens paper and xylol. Alcohol should not be used as it dissolves
the cement that binds the lenses. Set up the microscope in a convenient position, facing
the source of light. Place the object on the stage and adjust the mirror to the illuminant.
Always try to focus the blue sky if artificial light is not available. Direct sunlight should
not fall one the mirror. For prolonged work it is advisable to use artificial light of a
particular wave length.
Adjust the various parts as follows:
1. For Unstained Preparations :
a) Lower the condenser.
b) Close the iris diaphragm
c) Use concave mirror.
d) Focus under low power and then turn on to high dry power.
2. For stained preparations :
a) Lower the condenser.
b) Use concave Mirror.
c) Adjust the iris diaphragm to give an even illumination of the microscopic
3. For oil immersion examinations :
a) Raise the condenser completely.
b) Open the iris diaphragm.
c) Use plane mirror.
d) Study the object under low power first and then place a drop of the cedar
wood oil on the object and turn on the oil immersion lens. Rack the
objectives down until the tip of the objectives dips in the oil. Then using the
fine adjustment focus the object.
e) After the use remove the oil from the objectives and the object with a cloth
dipped in xylol.
f) Turn the nose piece until the low power objectives is in position.
1. Simple eye pieces with an eye lens at one end and field lens at the other.
The latter collects the image and throws into the focal length of the eye lens
in order to get a virtual image of the object.
2. Demonstration eye pieces in which a small hair or bristle is incorporated
which acts a pointer, and it is used to point out particular cells in the field.
3. Double Demonstration eye pieces: In this, ordinary eye piece is attached
with a side tube at the end of which there us another lens. Parts of the rays
of light that fall on the eye piece are diverted through a prism into the side
tube, so that another observer can see the field at the same time. The side
tube is provided with an adjustable pointer.
4. Micrometer eye piece: In this, a small, circular glass disc on which
graduations in microns is made is kept between the eye lens and the field
lens. The size of the object can be measured directly using this scale.
OTHER TYPES OF MICROSCOPES
I. DARK GROUND MICROSCOPE:
PRINCIPLE: The light is allowed to fall on the object in an oblique direction so
that it gets reflected from the object and thus enters and objective.
This microscope is used to study delicate organisms like spirochetes which are
not visualized by the ordinary compound microscope. It has special types of
condenser which does not allow the light to pass through the central portion
but the light is reflected with the help of the mirrored surface and emitted out
at an objective angle. Where there is no object in their path these rays pass out
of the objective. If a reflective object is introduced, viz., spirochetes, in their
path, those rays that are reflected by the object only will pass into the objective
and thus they are seen as bright objects against dark back ground.
USE: To demonstrate spirochetes.
II. PHASE CONTRAST MICROSCOPE:
PRINCIPLE: Light is assumed to be due to wave motion, to the upward and
downward vibration of other particles. They move at right angle to the direction
of the light. Since there is supposed to be a homogenous medium, the particles
can move in any direction at right angles to the path of the ray. But when the
light rays pass through a Nicol’s prism it becomes polarized and the direction
in which the particles move is known as the ‘optical path’. Now if another
prism is introduced in the path of the ray, it will pass unchanged provided the
optical path of the second prism is in alignment with the former and if the
second prism is rotated through 900, so that the optical paths are crossed, the
ray will be totally reflected out of the second prism. Thus no light will pass
through crossed prisms. In the polarizing microscope two prisms are used, one
below the condenser and the other above the objective. When both are crossed
no light will pass through the upper. Now, if a birefringent object, such as
cholesterol crystals, be placed at the object plane, those light rays that rays
that pass through these crystals will pass through the upper prism because
the prisms are no longer crossed from these rays. The rays that do not pass
through these crystals are absorbed by the upper prism. Thus the crystals will
be seen as bright object against a black background.
USE: To visualize the birefringent objects such as cholesterol crystals in
III. POLARISING MICROSCOPE :
PRINCIPLE: Light waves differ from one another in their (1) amplitude (2) wave
length & (3) phase. The phase difference cannot be made out by the eye. If by
some means, the phase difference could be converted into difference of
amplitude, different tissue components in an unstained preparation could be
made to appear of different degrees of brightness and so be distinguished easily
from one another. This aim may be achieved in a phase contrast microscope by
introducing a phase retarding a phase retarding plate in the objective.
USE: To study the living tissue in an unstained preparation.
IV. FLOURESCENT MICROSCOPE :
PRINCIPLE: Since the visible light is too great a probe to see minute particles,
ultraviolet rays which have shorter wave-length are utilized in this microscope.
The resolution power of this microscope will be accordingly increased. But
since the ultraviolet rays are not visible to the human eyes, fluorescent screen
is placed at the eye piece, which when struck by ultraviolet rays will glow and
thus the magnified image of the object is visualized.
The resolution power of this microscope will be between 0.1u, and 0.2u.
USE: To study inherent or induced fluorescence in tissues for various clinical
V. ELECTRON MICROSCOPE:
Since the light microscope have a limited resolution power, greater
magnifications to yield more cell details are not possible with these
microscopes. The electron microscope uses the electron as the probe, which
has a very small wavelength, and thus the resolution power of the microscope
will be greatly increased.
Construction: Here the lenses are made up of electromagnetic fields
whose strengths can be varied. The object to be examined should be ultra-thin,
since electrons have a poor penetration power. The electrons that are generated
at the tip of the cathode tube will be converged by means of an electromagnetic
field (condenser) on the object. The objective E.M. field will produce an
enlarged image of the object, which is either photographed or visualized by
using a fluorescent screen. Those parts of the tissue that are electron dense,
will allow less number of electrons to pass through and correspondingly a less
change in the photo graphic plate and vice-versa. When the positive print of the
photographic plate is made, the electron denser objects appear as dark bodies
and electron-rare objects as lighter objects.
USE: Since the resolution power of the microscope is extremely great,
ultra microscopic objects like viruses can be seen with great ease. The cellular
structures are also studied in greater detail in infections and neoplastic
RAPID DIAGNOSTIC METHOD
The clinical significance of many micro organisms has urged scientist to find out the
accurate and rapid diagnostic methods to identity them.
The rapid diagnostic method must be accurate, simple, quick and affordable for the
population for which it is needed.
It must also provide a result in time to institute effective control measures and
The rapid diagnostic method tests are
1) Urine reagent strip test – for Glucose, Ketone, Blood, Protein, PH & Urobilinogen
2) Pregnancy test – HCG, Combo, Urine/serum rapid strip test.
3) Blood Glucose Level – Glucometer
4) Infection disease – a) HIV rapid serum cord test
b) Dengue NSI rapid strip test
c) Malaria rapid strip test
d) HBV & HCV rapid test serum rapicord Etc…
An autoanalyser is an instruments/machine with which a large number of samples can
be analysed daily by partial or complete automation.
Automated syrings deliver fixed quantities of raegents to a series of samples or aliquotes.
Dilution, dialysis, separation of interfering substances and also treatment with
appropriate reagents to bring about the colour reaction are all done by the machine.The
colorimetric measurements are also carrried out automatically and the results are
recorded in a computer.
(a) Large number of samples can be examined on a very short period.
(b) Personal errors are minimised.
(c) A wide number of estimation can be done with one instrument.
(d) Small quantity of sample is required.
(e) Most suitable for laboratories handling large number of samples daily.
(a) The cost of the equipment is high, so not suitable for small or medium sized
laboratories handling a limited number of samples .
(b) Skilled persons are required to operate the machine.
MAXWELL M.WINTROBE (1901-1986)
Maxwell Wintrobe is a name almost synonymous with hematology. He has
taught, diagnosed, researched and wrote about hematological disorders for over
five decades. His name goes with the tube used for finding out erythrocyte
sedimentation rate. He was born in Halifax, Canada & trained in Canada and the
United States. He has served as a faculty member in prestigious institutes such as
John Hopkins University, University of Utah and Salt Lake County General
Hospital. He was also one of the first to pioneer treatment of cancer through
METHODS OF COLLECTION OF BLOOD
The blood should preferably be collected early in the morning before the patient has
eaten, and stored in a clean and sterile container in order to avoid bacterial
A. Capillary Blood: It is collected by pricking the finger tip or ear lobe in adults and
heel or great toe in children. Sterile needles, disposable lancets, spring lancet, B.P.
knife blades or capillary glass pricker can be used for pricking. The part should be
sterilized with spirit and the surrounding area gently pressed to produce
venostasis. A free flowing, large drop of blood should be obtained.
The capillary blood is used for the following tests:
1. Estimation of Hb% and R.B.C., W.B.C and Platelet counts;
2. Preparation of peripheral smears.
3. Blood grouping.
4. Estimation of blood glucose level by Somogyi’s micro-method.
B. Venous Blood: This is collected by using a dry, sterile syringe and needle. Anti
cubital vein in adults and external jugular vein in children are the usual sites of
veni-puncture. Blood should be drawn. In order to minimize haemolysis, a clean
puncture is essential. Blood should be stored in a chemically clean container with
or without the anticoagulant as the case may be.
Venous blood in necessary for the following tests:
1. Estimation of E.S.R., P,C,V. etc.,
2. Estimation of blood constituents like sugar, urea etc;
3. Bacteriological and serological examinations;
4. Blood grouping and cross matching.
C. Arterial Blood: Sometimes it may be impossible to collect blood from veins. In
such case arterial punctures can be attempted. Brachial artery and radial artery
are the usual sites. Sometimes arterial blood gives positive culture results when
venous blood is negative in case of sub acute bacterial endocarditic. Arterial blood
is collected most importantly for blood gas level studies.
The blood has to be kept in the fluid state for many of the hematological and
chemical examinations. In order to achieve this, anticoagulants have to be added in
appropriate proportions. Following are the various anticoagulants commonly used:
A. Oxalates: These salts combine with the calcium in the blood to form insoluble
compounds and thereby deplete the blood of its calcium which is necessary for
the coagulation of blood.
1. Dried Potassium Oxalate: For 10 ml of blood, 1 ml of 2% solution of potassium
oxalate dried in a container for blood at 800 C.
Disadvantages-causes shrinkage and destruction of cells.
2. Lithium Oxalate: Used in case of estimation of blood constituents since is does
not introduce an element which is tested in the blood. 2 mg of dried salt is
sufficient for 1 ml. of blood.
3. Double Oxalate or Wintrobe’s Salt: Combination of Ammonium and Potassium
oxalates (1.2 g and 8 g of these salts respectively and distilled water 100 ml.) is
used. 1ml of this solution dried in a container at 800c is sufficient for 10ml of
Advantages: Ammonium oxalate causes swelling of the cells where as potassium
oxalate shrinks. The action of both these salts is counterbalanced and thereby the
cells retain their original shape and size
Uses: Used in the estimation of E.S.R by Wintrobe’s method and P.C.V.
4. Saturated Potassium Oxalate: It is a 20% soln. of Potassium oxalate. One drop of
the supernatant solution is sufficient for 10ml of blood. It mixes more readily with
blood than dry salt.
1. Sodium Citrate: 1 part of 3.8% solution of the salt and 9 parts of blood
mixed for coagulation studies. Used in blood transfusion as the salt is
relatively non toxic and is excreted by the kidneys or utilized by the body.
3.8% solution in 1:4 ration with blood is used in ESR (Westergren’s
2. Acid Citrate Dextrose Soln. (ACD): Erythrocytes are better preserved in
the ACD than in Tri-Sodium Citrate alone.
Tri-Sodium Citrate 1.32 g
Citric Acid 0.42 g
Dextrose 1.40 g
Dist. Water add to 100 ml
1 ml of ACD is sufficient for 4 ml of blood. This is used in blood transfusion.
C. Heparin: It has an affinity for blood proteins and acts as an antithrombin and
antithromboplastin. 1 mg of dry heparin or 1000 I.U. of liquid Heparin suffices for
10ml of blood. Used in haematocrit studies and blood transfusion especially
exchange and rapid transfusion as in case of thoracic surgery; E.S.R and fragility
tests. It keeps for only three days.
D. Ethylene Diamine Tetra Acetate (EDTA): Its di-sodium and di-potassium salts are
used. They act as chelating agent and thus separate the calcium ions from the blood.
Used for most of the hematological and chemical tests except for the estimation of
N.P.N and electrolytes.
DETERMINATION OF HAEMOGLOBIN PERCENTAGE’
(Sahli’s Hellige Method)
Consists of (1) Comparator-Which consists of a rack with standard fixed in
front of ground glass.
(2) A graduated tube with markings in gms (2-22gms) and in
(3) Haemoglobinometer Pipette – glass pipette with 20cu mm
Used to pipette blood for hemoglobin estimation.
For Hemoglobin estimation.
Normal Range - Males 14-16g%
Methods used for Hb estimation
Sahli’s Acid Haematin Method
Procedure: 20µ 1(0.02ml) of blood is placed in a specially calibrated tube
containing N/10 HCI, to the 20 mark. Let the mixture stand at room temperature
for 10 minutes. During which time haemoglobin is converted to acid haematin.
The solution is diluted with distilled water until the colour matches exactly with
the standard of the comparator block. The percentage of Hemoglobin is read
directly from the calibration on the tube in which the solution is diluted.
Advantages: (1) Simple Method
(2) Small quantity of blood is needed.
(3) No sophisticated equipment is needed.
(4) Can be repeated often
Disadvantages: (1) Visual Error
(2) Other forms of Hb cannot be estimated
(3) Fading of the standard-false reading
(4) Time for 100% conversion takes 30 minutes.
(5) Affected by hyperbillirubinemia.
1) Alkali haematin method
2) Hb electrophoresis
3) Photo – electric colorimeter method – using cyanmethaemoglobin.
4) Spectroscopic method.
Conditions in which Hemoglobin is increased
1) Polycythemia Vera
2) Polycythemia secondary to hypoxia dehydration
Conditions in which Hemoglobin is decreased
2) Haemorrhagic disorders
The Colour Index
The colour index compares the mean haemoglobin content of one erythrocyte with
that of a normal erythrocyte which is taken as unit. In a normal individual both
haemoglobin level and R.B.C count are 100% of normal. Therefore the colour index is
1.0. A positive colour index greater than 1.0 indicates that each erythrocyte contains
more than the normal amount of haemoglobin, and a negative colour index less than 1.0
indicates lowered heamoglobin content per cell.
Colour Index = Hb% of normal
Erythrocytes% of normal
Normal Hb% - 14.5g
Normal R. B.C Count – 5 millions / cu mm
Calculation of ‘absolute values’ for mean cell size, Heamoglobin concentration:
1. Mean corpuscular haemoglobin gives the average weight of haemoglobin in each
red cell in picograms
M.C.H. = Hb grams per 1000ml of blood
R.B.C. count millions per cu. mm of blood
Normal Value: 27 to 32 pg (Picogram)
(MCH: decreased in iron deficiency anemia)
2. Mean Corpuscular volume is the mean value for the corpuscular volume as
determined by the haematocrit value and is recorded in Femto litres.
M.C.V. = Volume of packed erythrocytes per 1000 ml
R.B.C.count millions per cu.mm of blood
Normal Value: 80 to 94 fl (Femto liters)
MCV: Increased in all macrocytic anaemias and decreased in hypochromic anaemias.
3. Mean corpuscular haemoglobin concentrations given the average concentration of
haemoglobin in the erythrocyte expressed in percentage.
M.C.H.C. = Hb grams per 1000ml of blood x 100
Volume of packed R.B.C per 100 ml of blood
Normal Value: 28 – 32%
MCHC: is either normal of decreased but not increased.
ESTIMATION OF ERYTHROCYTE SEDIMENTATION RATE (ESR)
WINTROBE OR HAEMATOCRIT TUBE
It is a thick walled tube with narrow lumen, one end of the tube is closed. Total
length is 11 cm, diameter 2.5 mm. The tube is graduated from 0-100 mm. Markings on
one side are above downward and on other side below upwards. The below upward
markings are used for haematocrit and above downward for ESR.
Anticoagulant used is double oxalate, dry ammonium oxalate and potassium oxalate
Special pipette, Wintrobe’s pipette or Pasteur pipette.
Procedure for Haematocrit / PCV: Blood is filled in the pipette and tip of the pipette is
introduced into haematocrit tube touching the bottom and blood is slowly released in the
tube, no air bubble should be allowed to remain in the tube. Tube should be filled up to
the upper mark. This is centrifuged for 30 mins at 3000 revolutions per min and column
of packed RBC is recorded.
1) For PCV
A. Wintrobe’s Method of ESR
The E.S.R. denotes the velocity of sedimentation of R.B.C. per unit of time and is
expressed in mm at the end of one hour.
1. Wintrobe’s Method: Deliver 5 ml of venous blood into a tube containing 10 mg of
dry potassium and ammonium oxalate. Mix the samples well and fill a Wintrobe’s
haematocrit tube to the 100 mm mark by means of a capillary pipette. Place the
tube in exactly vertical position and observe the point on the scale to which the
red cells fall during one hour = ESR. Centrifuge the tube for 30 minutes at 3000
r.p.m. and read the volume of packed cells. Correct the ESR for anemia using
Normal Range: Men : 0 to 6.5 mm in one hour
Women : 0 to 15 mm in one hour
B. Different layers observed in Haematocrit Tube
1. Uppermost clear fluid i.e. plasma layer-colour is yellow in jaundice – brown in
intravascular haemolysis – milky in the case of increased lipids.
2. Just below it is thin layer of platelets.
3. Next to platelet layer which looks buffy is WBC.
The thickness of buffy coat gives an idea of WBC (1 MM = 1000/c.mm.). Buffy
coat smear is diagnostic in:
1) Aleukemic Leukemia
2) Kala – Azar and Trypanosomiasis
3) LE Cells
5) Nucleated RBC
Colour of the Plasma
1. Orange, green or yellow colour suggest-increased bilirubin
2. Pink or red suggest – Haemolysis
3. Cloudy / Opalescent suggest – nephrosis or abnormal hyperglobulinemia
C. Westergren’s Pipette
1. Length of the pipette – 300 mm (30 cm open at both ends. The lower 20 cms are
marked from 0 – 200mm. Diameter – 2.5mm.
2. Uses: 1. Estimation of ESR
3. Anticoagulants Used: 1) Sodium citrate 3.8% 0.4ml with 1.6 ml of venous blood.
2) If EDTA is used, the blood must be diluted with
trisodium citrate prior to testing (4 volumes to
4. Procedure: the anticoagulated blood is sucked up to the mark 0 and placed
vertically in a Westergren’ stand. The reading is taken of the end of the column of
sedimenting red cells.
Male 3-5 mm/1 hour
Women 4-7 mm/1 hour
Most sensitive ESR method for serial study of chronic diseases e.g tuberculosis.
1. Large amount of blood is needed.
2. Involves dilution when collected in EDTA.
1. The tube should be grease free.
2. Vertically placed in the rack.
3. Should be placed on a non-vibrant surface.
4. Should not be exposed to sunlight and heat.
Diseases in which ESR is raised:
2. Multiple myeloma
3. Rheumatoid arthritis
4. Collagen vascular diseases
6. Renal insufficiency
Diseases in which ESR is decreased: Polycythemia
Normal PCV : 45% + 2
Children : 35 – 47%
Other Method : Microhaematocrit method – Heparinised capillary tube
centrifuged and compared with the graph.
PCV is used to estimate severity of anemia and to calculate absolute value to classify
anaemia as microcytic, macrocytic and normocytic.
PCV is decreased in : Anaemias
PCV is increased in : Polycythemia
THE RETICULOCYTE COUNT
Brilliant cresyl blue. – 0.15 g.
Citrate saline solution -100 ml.
(1 part of 3% sodium citrate + 4 parts of 0.85 sodium chloride).
BrillIant cresyl blue in citrate saline solution is delivered in 1 ml amounts into 80
x 1 mm tubes (Kahn Tubes). Two to three drops of blood are added to each and mixed
with the diluent. The tubes are corked and allowed to stand for 10 to 15 minutes. Then
the tubes are centrifuged for 2 minutes, at 1000 revolutions per minute. The supernatant
fluid is removed by pipette leaving a volume of fluid about twice the volume of cells
below. The cells and supernatant fluid are well mixed and a drop taken on a clean slide
and a thin film is drawn.
Number of reticulocytes per 1000 R.B.C’s may be counted in the unfixed film or
after fixation in methyl alcohol for 3 minutes and subsequent staining with 1% aqueous
methylene blue for 30 seconds. Alternatively the blood films may be stained by
Leishman’s method. This is done if permanent preparation is necessary.
To facilitate counting, a circular piece of thin card board or paper with a square
slit cut in the centre may be attached to the eye piece.
Count about 500 cells in the unfixed film and calculate the percentage of
reticulocytes in the given sample of blood.
Infants at birth 2.0-6%
Children upto 5 years 0.2-5%
Interpretation: Reticulocyte counts are low in ineffective erythropoiesis
e.g.,myseloscelerosis, aplastic anemia, megaloblastic anaemia, thalassemia,
erythroleukemia and sideroblastic anaemia. Reticulocytosis occurs after blood loss or
effective therapy for certain kinds of anaemia, e.g., therapy of iron deficiency or
megaloblastic marcocytic anaemias. Reticylocytosis occours in hemolytic anaemias.
An increase of 15% to 20% of reticulocyte count is considered as index of
TOTAL RED CELL COUNT
a) R.B.C. Pipette
1. Consists of a pipette with bulb & a red bead in it. Red bead helps in mixing.
2. Markings on the pipette are 0.5 and 1.0.
Marking on the stem above bulb is 101.
3. Rubber tubing attached to the pipette should be thick walled to resist collapse
during suction & should be long enough (at least 10 inches) to permit easy
4. Blood is drawn upto 0.5 mark and R.B.C diluting fluid up to 101 marks. (1 in
5. Constituents of R.B.C. diluting fluid.
a) Dacie’s Formal Citrate
40% formalin – 10ml made upto 1
liter with 32g/l trisodium citrate.
Formalin acts as a preservative. In
Case of auto – immune hemolytic
anaemia, only trisodium citrate 32
g/l is used. (Formalin is not used
as it prevents the clumps of
agglutinated red cells from
b) Hayem’s Fluid
Mercuric chloride 0.5gm
Sodium chloride 1.0gm
Sodium sulphate 5.0gm
DH2O to 200ml
The other diluting fluids used are as
c) Sodium Citrate 3.0gm
d) Toisson’s Fluid
e) Gover’s Solution
Filling the Pipette: The pipette must be clean and dry. Suck the blood to mark 0.5
holding the same almost horizontally. If the blood goes slightly beyond the mark, draw it
back by touching the tip of the pipettes to a moistened towe. Quickly wipe off the blood
adhering to the tip, plunge it into the diluting fluid and suck the fluid up to the mark
101, slightly rotating the pipette meanwhile. Close the ends of the pipette with the
fingers, and shake vigorously for about a minute or two.
Charging the Counting Chamber: Clean the counting chamber and cover glass. Adjust
the cover glass properly. Shake the pipette well. Now quickly blow 2-3 drops of fluid and
wipe them off. Holding the pipette in an inclined position, touch the tip of the pipette to
the angle between the edge of the cover glass and one of the floor piece. The fluid must
nearly or entirely fill the space beneath the cover glass, none into the depression and
there must be no air bubbles.
Counting of Erythrocytes: Allow atleast 5 minutes for the red cells to settle. The RBC’s
must be evenly distributed over the whole disk. Count the erythrocytes in 10 small
squares under the dry high power objective. Count the cells which touch the lower and
left sides and omit those touching the upper and right sides.
Calculation: In the Neubauer counting chamber each small square is 1/20 mm, on each
side, the depth of the chamber is 1/10mm, the dilution is 1:200 and when 80 squares
are counted the number of erythrocytes per c.mm, is calculated from the formula: (1/20
x 1/20 x 1/10 x 1/200 x 80) = 10,000 i.e., multiply the number of cells in 80 squares by
Sources of error: (1) Inaccurate dilutions; (2) Slow manipulation, allowing some blood to
coagulate in the capillary portion of the pipette; (3) Imperfect application of the cover
glass; (4) Uneven distribution of erythrocytes and presence of air bubbles; (5) Counting
insufficient number of cells (less than 500) ; (6) Personal bias in counting.
1. Counting chamber is “improved Neubauer’s.
2. Consists of a thick rectangular slide which has a central H shaped depression on
either side of which are two stages. Each stage consists of a ruled area of 9 sq mm
divided into 9 squares. The central square is further divided into 25 small squares
by triple lines. Each of these 25 squares is further divided into 16 squares. Depth
between cover slip and ruled area – 0.1 mm.
Improved Neubauer counting chamber (above) and as it is seen under low
power objective (below)
a) Blood and corresponding diluting fluids drawn in the pipettes.
b) First 3-4 drops from the corresponding pipettes discarded.
c) Cover slip placed over Haemocytometer.
d) Pipette held at an angle of 350 and the tip should touch the groove at the edge
of cover slip.
e) Fluid runs under the cover slip by capillary action- till the stages are covered
f) Cells in the chamber to settle for several minutes.
g) Counting done with condenser diaphragm of microscope partially closed to
make the cells stand out clearly.
h) Calculation – Number of cells counted are multiplied by a factor i.e.,
W.B.C.Count - W x 50
R.B.C.Count - R x 10000
Platelet Count - P x 1000
1. Due to nature of sample – coagulated blood
2. Operator’s error – faulty technique
3. Errors due to equipment – inaccuracies in graduations.
4. Inherent or field error.
1. RBC Count- Central square- 5 big squares in centers with small squares.
2. WBC Count – corner square – (4)
3. Absolute eosinophil count – all central and corner squares.(9)
4. Cell counts of cerebrospinal fluid and other body fluids- all central and
5. Semen analysis – count-corner squares.(4)
6. Platelet Count – 25 big squares in center with 16 small squares each.
R.B.C Count Adult Male 4.5 – 6.2 millions/µ1
Female 4.0 – 5.5 millions/µ1
Increased RBC Level Decreased Level
a) Age- at birth more a) Old age
b) Cases of Haemoconcentration b) Anaemias
c) Central cyanotic states c) A plastic Anemia
d) Polcythemia Vera
THE TOTAL LEUKOCYTE COUNT
Instruments used: W.B.C.PIPETTE
1. It contains of a pipette with small bulb with a white bead in it. White bead helps in
2. Markings on the pipette are 0.5 and 1. Marking on the stem above bulb 11.
3. Rubber tubing attached to the pipette – heavy
walled to resist collapse during suction and
should be long (at least 10 inches) to permit
4. Blood is drawn into 0.5 mark and WBC diluting
fluid upto 11 mark (1 in to 20 dilutions).
5. WBC Diluting Fluid
Glacial acetic acid – 1.5 ml.
1% aqueous Gentian Violet – 1 ml.
Distilled water – 98 ml.
Acetic acid lyses erythrocytes, Gentian violet stains
Draw blood into the WBC pipette to the Mark
0.5.Suck the diluting fluid to the mark 11 and
mix at least for 2 minutes by tilting and rotation.
Charge the counting chamber with the fluid
and allow to settle. Using 1/6 objective,
count in four 1 mm. square Areas.
Take usual precaution as in R.B.C. Count.
Let the No. of cells counted in 4 WBC Squares = X cells
Volume of 1 large square = 1 x 1 x 1 c.mm.
4 large squares = 4 x 1 = 2 c.mm.
2/5 c.mm contain – X cells.
1c.mm. =X x 5/2 cells
Dilution = 1:20
=X x 5 x 20/2=50 x X cells
Total WB.C.COUNT = X x 50 cells/c.mm.
ABSOLUTE EOSINOPHIL COUNT
Increased eosinophil count is often associated with allergic reactions, parasitic infections,
brucellosis and in certain leukemias. Increase in the adrenal function (hyperadrenalism
or Cushing’s syndrome) is associated with a fall in eosinophil count.
40-440/cu mm (µ I)
EDTA or heparinized blood
Blood is diluted with a special diluting fluid, which removes red cells and stains the
eosinophils red. These cells are then counted under low power (10 X) in a known volume
of fluid by using Neubauer counting chamber.
2) Improved Neubauer chamber of Fuch-Rosenthal counting chamber
3) Diluting fluid: (Hingleman’s solution)
It is prepared as follows:
(a) Yellow eosin : 0.5 g
(b) 95% phenol : 0.5 ml
(c) Formalin : 0.5 ml
(d) Distilled water : 99 ml
1) Pipette 0.36 ml of diluting fluid in a test tube.
2) Add 0.04 ml of blood (use Hb pipette, twice).
3) Mix and keep for 10 minutes
4) Mix the diluent and charge the counting chamber,
5) Let is stand under a moist petri dish for about 2 to 3 minutes.
6) Count the cells under low power objective in all nine squares of Neubauer
Total number of eosinophils , cu mm (µ I)
Number of cells counted X 10
a) Dilution = 10
b) Volume of fluid = area counted X depth
= 9 sq. mm X 0.1
MAKING OF BLOOD FILMS
Thin Film: Clean the finger tip. After allowing the skin to dry, puncture it with a
sterile blood lancet or needle with a firm quick stab. Wipe away the first drop which
appears. Take a small drop of blood a clean slide about ½” from the end, taking care that
the slide does not touch the skin. Place the edge of a second slide against the first slide
at an angle of about 350 and draw it up against the blood drop which will immediately
run across the end filling the angle between the slides. Push the upper slide back along
the other slowly to get a thin film. Dry the blood film in air.
Thick Film: Take 2 to 3 drops of blood close together on the slide, spread them out
enough to show hands on watch through the film. Dry in air for ½ to 1 hour.
Fixation: Blood films are dried rapidly in air and fixed by immersing in methyl alcohol for
3 minutes or in ethyl alcohol for 5 minutes.
Staining Blood Films: The Romanovsky stains depend for their action on the compounds
formed by the intersection of methylene blue and eosin. The stain gives a reddish purple
colour to the chromatin of malaria and other parasites. This colour is due to substance
which forms when methylene blue is ripened either by age as in polychrome methylene
blue or by heating with sodium carbonate. There are several modifications of the original
Romanovsky stains, of which well-known are LEISHMAN’S AND GIEMSA’S STAINS.
I. Leishman’s Stain :
(a) Pour the undiluted stain on a dry, unfixed blood film. Allow 3 minutes. Methyl
alcohol in the Lieshman’s stain fixes the film.
(b) By means of pipette with rubber teat add buffer solution equal in quantity to
the stain added. Mix by gentle blowing. Allow seven minutes.
c) Wash the film in distilled water allowing the preparation to differentiate until
the film appears bright pink in colour about half a minute.
(d) Dry the film in air.
II.Giemsa’s Stain: (1) Rapid method: Fix the blood film in methyl alcohol for 3
minutes. Pour the diluted stain (1:2) and allow to act for 3 minutes. Wash with water for
a minute. Dry in air.
(2) Slow Method: Fix the film in methyl alcohol for 3 minutes. Take a dish with diluted
stain (1:10).Place a piece of glass rod in it.
Lay the slide film downwards in the fluid with one end of the slide resting on the rod.
After 24 hours wash the slide and dry. This particular method is also useful in
Differential count: Count the cells as arrows indicate the directors of movement of the
Note: As the larger leukocytes, particularly granulocytes have a tendency to be more
abundant at the margin and at the end of the smear, while counting use Turrel’s method.
Normal Leukocytes Percentage Abnormal Leukocytes
Polymorphs : 65% to 75% Myeloblasts Promyelocyte
Eosinophils : 2% to 5% Myelocytes
Basophils : 0.5% to 1% Metamyelocytes
Lymphocytes: 20% to 25% Lymphoblasts
Monocytes : 3% to 8% Monoblasts
Exercise: Draw a thin film of your blood. Stain the film with Leishman’s stain. Make the
following counts under oil immersion objective
STUDY OF LEUKOCYTES
I. Granulocytic Series:
1. Myeloblast: 15-20µ in diameter. The nucleus is round or oval, eccentrically
placed with a delicate network of chromatin containing 2 to 3 nucleoli. The
cytoplasm is abundant, non-granular and basophilic.
1. Myelocyte: 12 to 20µ in diameter. The nucleus is large, round or oval but is
frequently flattened on the medial side. Nucleoli are absent and the chromatin is
coarser. The myelocytes are classified according to the medial side. Nucleoli are
absent and the chromatin is coarse. The myelocytes are classified according to the
colour of the granules in the cytoplasm they take with Leishman’s stain, into
neutrophilic, eosinophilic and basophilic myelocytes. The cytoplasm is less
basophilic and more extensive.
2. Neutrophilic metamyelocyte-Juvenile cell: 14 to 16 µ. The nucleus in smaller,
the strands of chromatin are dense and more deeply staining. The cytoplasm is
eosinophilic with neutrophilic granules.
3. Stab-cell Band neutrophils: 12 to 14 µ. The nucleus elongated and rounded at
the ends and curved. The chromatin forms a coarse network. The cytoplasm is
eosinophilic and contains fine dust like neutrophilic granules.
4. Segmented Neutrophils: 10 to 12 µ. The nucles is lobulated, having 2 to 5 lobes
of irregular size. The cytoplasm is eosinophilic with fine neutrophilic granules.
5. Eosinophil: 10 to 12 µ. The nucleus is bilobed, connected by a thin chromatin
thread. The coarse highly refrigent granules in the cytoplasm are eosinophilic.
6. Basophil: 9 to 10 µ. The nucleus is bilobed, elongated and usually bent in the
form of ‘S’. The granules in the cytoplasm are larger and take a strong basic stain.
As the granules are very coarse and numerous they may obscure the nucleus.
II. Non-Granulocytic Series:
1. Lymphoblast: (12 to 15 µ). The nucleus is large, round or oval with coarse
chromatin network. The cytoplasm is slightly basophilic. No granules. 1 or 2
nucleoli are present.
Lymphocytes: (6 to 10 µ). The nucleus is oval or round, dark blue with dense
chromatin network. The cytoplasm is slightly basophilic, abundant in the large
lymphocytes and scanty in the small lymphocytes.
2. Monoblasts:(6 to 10µ). The nucleus is large, has a fine chromatin network which
stains light purple and contains nucleoli. The cytoplasm is basophilic and darker
than that of the lymphoblasts.
Monocytes : ( 9 to 12µ). The nucleus is large, oval or kidney shaped. The
chromatin strands are fine and dense. The cytoplasm is slightly basophilic,
3. Plasma Cells :( 0 to 15µ). The nucleus is eccentrically situated. The cell is round or
oval with chromatin in the form of triangular clumps giving the nucleus a
‘cartwheel’ appearance. The cytoplasm is basophilic with a few acidophilic
granules. A clear crescentic zone,the para nuclear demilune can also be seen.
1. Acute :
2. Sub acute :
3. Chronic :
Aleukemic Leukemia :
BONE MARROW BIOPSY
A. For Diagnosis:
1. High colour index anaemias – (a) Megaloblastic anaemias marrow shows typical
megaloblastic red cell formation with the presence of giant metamyelocytes. Such
a picture occurs in pernicious anaemia, nutritional megaloblastic anaemia
usually secondary to steatorrhea ,megaloblastic anaemia of pregnancy and
megaloblastic anaemia due to anti-convulsant drugs, (b) hemolytic anaemia –
suggested by cellular normoblastic marrow. (c) Aplastic anaemia, few cells mostly
lymphocytes, monocytes and plasma cells. (d) Myxoedema and renal failure-
hypoplastic marrow with moderate diminution of all cell types.
2. Tropical diseases – (a) Kala-azar – L.D. bodies may be readily distinguished
within the monocytes. (b)Malaria-Marrow tissue for identifying the malaria
3. Aleukemic leukaemia – 50% or more cells are primitive leukocytes though
abnormal cells are absent or too few for diagnosis in peripheral blood.
4. Myelomatosis – infiltration with plasma cells.
6. Malignancy – at times, cells from malignant tumors occur in marrow smears.
7. Gaucher’s disease – reticulum cells stuffed with abnormal lipid.
B. For prognosis :
1. Primary form of thrombocytopenic purpura – if the marrow shows increased
activity of normoblasts and megakaryocytes. In this type, the response to therapy
is good but not in the secondary type.
2. Chronic myeloid leukemia- if myeloblasts are 10% or less, response to treatment
is likely to be good, if 50% or more the reverse.
CONTRAINDICATION - Hemophilia and allied disorders of coagulation.
Bone marrow study can be facilitated either by bone marrow aspiration are by biopsy
BONE MARROW NEEDLE
Parts- Wide Bore Needle
Types :( 1)Klima needle Aspiration
(3)Jamshidis needle-both aspiration and biopsy.
Salah’s needle Jamshidis needle
Dry sterilization in hot air ovens.
Site of BM Biopsy
1) In Infants – Tibial tuberosity
2) Grown up children and adults-Anterior superior iliac spine
Anterior Surface of sternal body membrane.
TECHNIQUE: (1) Marrow aspiration is done with marrow aspiration needle. The
skin, subcutaneous tissue and periosteum over the posterior iliac crest (or
manubrium sterni) are infiltrated with 2% procaine. The marrow needle is
pushed through the bone with a boring motion, the guard being kept at a
distance of about 1 cm above the surface of the skin. When the needle has
entered the marrow the sylette is withdrawn and a long 10 ml syringe
attached, 0.2 – 0.3 ml of marrow fluid is aspirated. The needle with trocar is
removed and the site of aspiration is sealed with benzoin tincture. Marrow
juice drawn off the smeared onto glass slides. In selected patients, residual
aspirate is placed in appropriate specimen containers for chromosomal
analysis, microbiological culture, cell culture and electron microscopy.
2) Marrow trephine – immediately on completion of the aspiration a
trephine biopsy (with a Jamshidi-Swaim needle) of an adjacent area of bone
is performed through the same puncture site. After expulsion of 2 cm core
of bone and its enclosed marrow from the needle, the biopsy specimen is
smeared gently across three glass slides and then placed in fixative for
subsequent histopathological processing and staining.
Preparations of bone marrow
smears - marrow material drawn off
are smeared onto glass slides and
extra material is collected in the
EDTA tube. The marrow practical are
picked by a forecep and put on glass
slides and crushed smears are
Bone marrow aspiration staining
Routine – Leishman & wrights stain
Special – Perl’s stain – Iron content
Bone Marrow biopsy
Routine – H & E – Stain
Special - Reticulin stain - Myeloscelrosis
Cause of ‘dry’ or bloody tap - 1. Acute myeloblastic or lymphoblastic leukaemia
2. Acute promyelocytic leukaemia.
3. Hairy’ Cell leukaemia.
5. Refractory anaemia with cellular marrow
6. Malignant infilitration of marrow
Normal Marrow- Nucleated cells 20,000 to 100,000 per c.mm predominant cells –
granulocytes of normal types, and a few normoblast, both are showing
some mitoses, megakaryocytes present, other cells few. No abnormal
ORGANISATION AND FUNCTIONING OF BLOOD BANK
The organisation of blood bank should receive utmost attention and a careful
design and management for smooth functioning of various components. The goal of
blood bank is to provide effective blood and blood components which are as safe as
possible and adequate to meet the patients need.
Registration and medical examination room for registration and selection of donors.
Blood collection room (Bleeding area) where blood is collected from the voluntary
and replacement blood donors.
Room for blood group serology (serology room) – The blood which is collected from
the donors and the recipient is tested for the blood groups and cross matching.
Room for testing communicable disease like hepatitis, syphilis, malaria, HIV
antibodies. The collected blood is tested for above mentioned antibodies. It should
have ELISA test kits with reader.
Component separation room - The blood bags which are tested negative for
communicable diseases are taken and components of blood like RBC & WBC,
platelets & plasma are separated in different blood bags and stored.
Storage room – The blood which is tested and separated is stored at temperature 4-
6 degree C0 with recording thermometer and alarm device.
Records room and store- Correct maintainance of record is very important in Blood
bank. Records like blood donor register, blood stock register, issue register, etc. are
Basic functions of blood bank.
Recruitment & retention of voluntary and replacement blood donors.
Collections, processing, storage and transportation of blood and blood components.
Laboratory procedures (investigations.)
Participating in clinical use of blood and blood components.
Teaching and training of personnel.
Research and developments.
EXERCISE-VISIT TO CENTRAL BLOOD BANK
DETERMINATION OF BLOOD GROUPS
Puncture a finger and collect 2 to 3 drops of blood in a concavity of a porcelain slab
carrying about 1 ml of 1% sodium citrate solution. Mix gently by means of pipette.
In a double paraffin ring slide, place one drop of ‘A’ serum in the centre of the ring
and a drop of ‘B’ serum in the centre of the other. Use separate pipettes for each
serum. Mark with glass pencil. Add one drop of corpuscle suspension to each and
mix by gently rocking the slide. Allow to stand for about 5 minutes, occasionally
rolling or tilting the slide to ensure thorough mixing. If definite agglutination has
occurred, a reading and report may be made. If there is no definite agglutination,
cover each ring with a cover glass and examine at the end of 30 minutes for final
reading. The readings are made as follows:
No agglutination by either A or B sera - Group ‘O’
Agglutination by both A and B sera - Group ‘AB’
Method of Cross Matching: Direct matching tests are always advisable before
transfusion to guard against the possibility of transfusing with incompatible sub-
groups [Sub –Groups A1, A2, A3, A2B and Rh).
In one ring of a double paraffin ring slide, mix a drop of donor’s serum with a drop
of suspension of recipient’s corpuscles. In the other ring mix a drop recipient’s
serum with a drop of suspension of donor’s corpuscles. After 15 minutes take the
Normal Values Conditions causing variations
1. Blood: Increased in : Decreased in:
Haemoglobin Men 13.5-18.0 Dehydration Anaemias
(gms.per 100 ml) Women 11.5-16.4 First two weeks of infancy Primary and secondary
Infants (full term cord Polycythaemia
Children, 1 year
Children, 10 year
Total Red Blood Cells Men 4.5-6.5 Polycythaemia Anaemias:
(Million per cubic Women 3.9-5.69 Dehydration, Anoxia Primary and secondary
Millimeter) Infants (full term cord Congestive cardiac failure
Blood) 4.0-6.0 Congential heart disease
Children, 1 year Congential heart disease
(mean) 4.5 High altitude
Children 10 years
Reticulocytes Adults 0.2-2.0% Perinicious anaemia Aplastic Anaemia
Infants (full term) 2-6% During treatment of
Blood: Increased in Decreased in:
Platelets 150,000-400,000 Hemorrhagic Thrombocytosis Pancytopenia (Hypers-
After Splenectomy plenism) Purpura
After exercise haemorrhagica
Erythrocyte Men 3-5 mm in 1 hour Many general diseases, Polycythemia
Sedimentation Rate 7-15 mm in 2 hours Febrile conditions (useful for
(Westergren Method) Women 4-7 mm in 1 hour prognosis in tuberculosis
Sedimentaion Rate 10-12 mm in 1 hour rheumatism &
Men 0-9 mm in 1 hour Coronary thrombosis
Women 0-20 mm in 1 hour
Bleeding Time 0-7 min. Essential and Symptomatic
(lvy’s Method) Thrombo-
state in infants.
Clotting time 5-11 min. Hemophilia, Christmas diseases,
(Lee & White Method) Anti Coagulant therapy.
Red Cell Osmotic No definite haemolysis at Spherocytosis Cooley’s and Sickle Cell
Fragility. Concentrations higher than anaemias: polycythemia
0.45% Nacl.Sol. Vera.
Complete haemolysis at
0.35% Nacl. Sol.
Packed Cell Volume Men 40-54 Dehydration, Burns Anaemia
(P.C.V) % Women 36-47 Polycythaemia
Infants (full term cord
Children, 1 year
Children, 10 year
Mean Corpuscular Adults 76-96 Pernicous & Aplastic Iron deficiency anaemia
Voiume (M.C.V) fl Anaemia, Liver Disease, Acholuric Jaundice
(femto liters) Sprue, Tropical Macrocytic Polycythaemia Vera
Mean Corpuscular Adults 27-32
Haemoglobin (M.C.H.) pg
Mean Corpuscular Adults 32-36% Normal in aplastic Iron deficiency anaemia
Haemoglobin Con- anaemias and pernicious Acholuric Jaundice and
centration (M.C.H.C.) anaemias. Anaemias.
Total White Cells Adults 5,000-10,000 Pyogenic infections Typhoid, some virus
(per cmm) Children 5,000-14,000 Leukamia, Infectious infections, kala-azar,
Mononucleosis, Whooping Chronic Malaria,
Cough, most allergic Splenic Anaemia,
STUDY OF ANAEMIA
MICROCYTIC HYPOCHROMIC ANAEMIA
Microcytic hypochromic cells
HISTORY: A 60 yr old male complains of weakness,fatigue and bleeding per
rectum(Haemorroids) since 8 months. Hb-4.8 gms/dl,TC-88OO cells/cumm,platelet-2.5
RBC : Predominantly microcytic hypochromic cells
WBC : Normal in count, morphology
PLATELET : Adequate
ABNORMAL CELLS: Nil
HAEMOPARASITE : Nil
IMPRESSION : MICROCYTIC HYPOCHROMIC ANAEMIA
NORMOCYTIC NORMOCHROMIC ANAEMIA
Normocytic normochromic cells
HISTORY: A 20 yr old male was admitted with a history of minor accident. O/E-
haematoma seen in the scalp.Hb-11gm/dl,RBC-2.9 millions, TC-5800cells,Platelet3 lakh.
RBC : Predominantly normocytic normochromic cells
WBC : Normal
PLATELET : Adequate
ABNORMAL CELLS: Nil
HAEMOPARASITE : Nil
IMPRESSION : NORMOCYTIC NORMOCHROMIC ANAEMIA
Normoblast Microcytic hypochromic cell Target cell
Fragmented RBC Lymphocyte
Platelet Basophilic stippling
HISTORY: A 4 yr old child is suffering from growth retardation, irritability and
anorexia till infancy. O/E, pallor-marked, huge enlargement of liver and spleen with
sternal tenderness, depressed nasal bridge with malar prominence.Hb-6gm%.
RBC : Moderate anisopoikilocytosis, predominantly microcytic hypochromic
cells along with target cells and fragmented RBC and normoblast
WBC : Normal
PLATELET : Normal
ABNORMAL CELLS: Nil
HAEMOPARASITE : Nil
IMPRESSION : THALASSEMIA
Platelet Polychromatophilic RBC
HISTORY: 34 year old male c/o slight pallor, jaundice, and sclera shows icterus. History
of cholecystectomy five years back. O/E Spleen palpable 3 cm below the left costal margin.
RBC : Normocytic Normocromic, spherocytes and
WBC : Normal
PLATELET : Normal
ABNORMAL CELLS: Nil
HAEMOPARASITE : Nil
IMPRESSION : SPHEROCYTIC ANAEMIA
HAEMOLYTIC ANAEMIA WITH G6PD DEFICIENCY
Platelet Bite cells microspherocyte
Polychromaticphilic RBC Fragmented RBC
HISTORY: A 57 yr old male presents with passage of dark,smoky urine for
3 days. He has taken primaquine 2 days back. O/E, pale with jaundiced skin and
scleral icterus. Moderate splenomegaly and hepatomegaly.
RBC : Normochromic normocytic cells with prominent polchromas
ia Bite cells, microspherocytes and fragmented cells seen.
WBC : Normal
PLATELET : Adequate
ABNORMAL CELLS: Nil
HAEMOPARASITE : Nil
IMPRESSION : HAEMOLYTIIC ANAEMIA WITH G6PD deficiency
HISTORY: A 60 yr old male admitted to the hospital with difficulty in breathing with
past history of bronchial asthma.
RBC : Are normocytic normochromic
WBC : Normal in count with increase in eosinophil count
PLATELET : Adequate.
ABNORMAL CELLS: Nil
HAEMOPARASITE : Nil
IMPRESSION : EOSINOPHILIA.
STUDY OF LEUKAEMIAS
ACUTE MYELOBLASTIC LEUKEMIA
Myeloblast with auer rod Myeloblast Nucleoli
HISTORY: A 23 yr old male presents with bleeding gums for 5 days .O/E: Pallor,
splenomegaly and gingival hyperplasia,petechial bleeding spots over trunk is seen.
RBC : Normocytic normochromic
WBC : Myeloblast seen with auer rod
PLATELET : Decreased
ABNORMAL CELLS: Myeloblasts
HAEMOPARASITE : Nil
IMPRESSION : ACUTE MYELOBLASTIC LEUKEMIA
CHRONIC MYELOID LEUKEMIA
HISTORY: A 57 yr old male complaining of progressive weakness,weight loss and
anorexia for last 3 months.O/E-moderate pallor with massive splenomegaly.Hb-
10gm/dl,TC-2-3 lakh/cumm.,platelet 3 lakh
RBC : Normochromic normocytic cells with presence of normoblasts
WBC : Very high count, mature and few immature granulocytes
seen. Basophil count is increased
PLATELET : Adequate
ABNORMAL CELLS: Granulocytes
HAEMOPARASITE : Nil
IMPRESSION : CHRONIC MYELOID LEUKEMIA
Abnormal Plasma cell
HISTORY: 50 year old lady came to the hospital with c/o of fractured rib, O/E there is
pain and mild swelling, X-ray showed pathological facture of 4th rib with osteolytic lesion
RBC : Normocytic normochromic
WBC : Normal
PLATELET : Normal
ABNORMAL CELLS : Plasma cells
HAEMOPARASITE : Nil
IMPRESSION : MULTIPLE MYELOMA
STUDY OF MICROFILARIA
HISTORY: 40 year male came to hospital with the c/o of swelling and pain in left leg. On
examination there is massive swelling (elephantiasis). Peripheral smear showed above
RBC : Normocytic normchromic cells
WBC : Normal
PLATELET : Normal
ABNORMAL CELLS: Nil
HAEMOPARASITE : Microfilariae
IMPRESSION : MICROFILARIAE
STUDY OF MALARIAL PARASITE
HISTORY : A16yr old boy is admitted into medicine ward with irregular pyrexia for
more than 3 months/E-Pallor-moderate, Spleen-moderatly enlarged,firm ,Liver-mild
RBC : Normocytic normochromic cells
WBC : Normal in count and morphology
PLATELET : Decreased
ABNORMAL CELLS: Nil
HAEMOPARASITE : Malarial gametocyte seen
IMPRESSION : MALARIAL PARASITE
WILLIAM BOYD (1885 – 1979)
William Boyd trained in medicine in Edinburgh and was Professor of pathology
successively in the universities of Manitoba, Toronto and British Columbia. He wrote
textbooks which related clinical; disease to autopsy findings in lucid readable English.
These books were popular worldwide as undergraduate and Post- graduate medical texts,
and helped to give the Manitoba Medical School an international reputation in the
1930’s. “It has become the fashion to regard morbid anatomy both gross and microscopic
as somewhat of an outworn creed, a science as dead as the material with which deals.
But morbid anatomy is not dead, and never has been except in the minds of those whose
dull minds would take the breath from the most vital subjects.” W.Boyd, a Textbook of
Pathology: an introduction to Medicine, Lea dn Febiger, Philadelphia, 1932.
EXAMINATION OF URINE
Urine should be examined soon after being passed. For routine examination random specimen is
satisfactory, but for certain investigations urine passed at a particular time is valuable.
Volume of Urine: A healthy adult excretes about 1000 to 1600 ml of urine 24 hours.
POLYURIA: Implies an increased volume of urine and occurs in (1) Increased ingestion of
fluid; (2) Diabetes mellitus; (3) Chronic renal diseases; (4) Diabetes insipidus and (5) Certain
OLIGURIA: Means a decreased urinary out-put (less than 500 ml/24 hrs) occurs (1)
Restricted intake of fluid; (2) Excessive loss of fluid through extra-renal channels e.g. sweating,
diarrhea, vomiting etc. (3) Reduced blood supplied to kidneys e.g., in hemorrhage, dehydration
and shock. (4) Renal diseases like acute glomerulo-nephrits, nephritic syndrome, acute tubular
necrosis such as in crush syndrome, incompatible transfusion, heavy metal poisoning,
sulphonamide,anuria etc, (5) Addison’s disease.
ANURIA: Means excessive suppression of urine formation due to severe impairement of
venous blood flow, obstruction to the outflow of urine or severe pathological change within the
nephron itself. In this condition the urine outflow will be less than 100 c.c. /24 hours.
Colour: Normal urine is pale-yellow of light-straw in colour due to the presence of urochrome,
the chief urinary pigment. In polyuria, urine almost becomes colourless. In oliguria, due to
concentration, the colour is dark brown. Presence of blood, melanin and abnormal pigments and
various drugs etc., may change the colour of urine.
Transparency and Turbidity: Normal urine is usually clean when passed-fresh, but
sometimes phosphates may produce turbidity in fresh urine. Urine may become turbid on
standing due to the presence of mucus or the formation of amorphous or crystalline deposits or
bacterial growth. Infections of bladder, prostate or urethra are sometimes associated with
increased secretion of mucus. Pathological causes of turbidity of urine include pus, blood or
bacteria which are identified by microscopy.
Odour: Normal fresh has either no smell or some characteristic aromatic odour. Ammonia
odour is detected in decomposed urine and smell of acetone in ketonuria.
Reaction of Urine: In health, the pH of urine varies from 4.85 to 8.0 but usually it is slightly acidic
(about pH 6).
Technique: The reaction of urine is determined with blue and red litmus paper (pH range 5.8
to 7.4). Alkaline urine turns red litmus paper blue and acid urine turns blue litmus paper red.
Both blue and red litmus papers turn reddish purple when the urine is faintly on the acid side of
neutral (pH 7.0). The urine when examined must be fresh as it turns alkaline on standing due to
The acidity of urine is decreased after a meal and with a vegetarian diet the reaction may become
alkaline. A strongly alkaline urine may also be due to infection with urea fermenting organisms.
Ingestion of citrates and bicarbonate tends to make urine alkaline.
The degree of acidity is increased in high protein diet, febrile illness, ketonuria and in leukaemia.
Urine may be made acid with ingestion of ammonium chloride, mandelic acid or ammonium
SPECIFIC GRAVITY URINOMETER
It is an instrument by which the specific gravity of urine is determined.
It is a hydrometer adapted to measure the specific gravity of urine room temperature. It is
a weighed glass cylinder, with a bulb containing mercury and a stem. The stem has a scale
with readings from 1,000 to 1060 with divisions of 0.001 to 0.002 used at temperature of
It floats in urine taken in the container.
The urinometer vessel is filled three fourths full with urine (minimum volume required is
15 ml). The urinometer is inserted with a spinning motion to make sure that it does not touch
the sides of the container and floats freely. It should also not touch the bottom of the cylinder.
The reading is taken at the bottom of the meniscus at eye level.
One must see that there is no surface bubble in the urine. The reading is corrected as
required for dilution, temperature and total protein.
In case of the test, temperature being more than 150C, for every 30C rise in
temperature, 0.001 is added to the reading and for each 30C below this, 0.001 is
subtracted from the reading.
(e) The urinometer should be checked every day by measuring specific gravity of distilled
water which has a specific gravity of 1.000. If the urinometer does not give a reading of
1.000 an appropriate correction must be applied to all readings taken with that
The accuracy of a urinometer may be further checked in solutions of known
Eg: Solution of potassium sulphate – specific gravity of 1.015.
(f) Normal and Abnormal values:
Normally the value ranges between 1.003 to 1.030. Substances which influence
specific gravity are urea sodium chloride, phosphates, albumin and sugar.
= Urine of low specific gravity is called”Hyposthenuric” sp.gr.is less than 1.003. eg.
= Urine of fixed specific gravity are called “lsosthenuric” sp.gr.is fixed at 1.010. eg. In
end stage renal disease.
= Urine of high specific gravity> 1.030 is seen in Diabetes mellitus, Nephrotic
The measurement of specific gravity gives an indication of urinary total solute
Several other methods used are:
2) Reagent strip method.
(FOR ABNORMAL CONSTITUENTS)
Proteinuria: It is not found in normal life although it is seen in some physiological
conditions like severe muscular exercise, orthostatic postural proteinuria, exposure to cold etc.
The pathological conditions due to pre – renal causes are toxicity etc. Those due to renal causes
are acute or subacute glomerulonephritis, nephrotic syndrome, destructive lesions like
tuberculosis of the kidney, neoplasms, calculi, nephrosclerosis etc. Those due to post-renal
causes are pyelitis, cystitis, urethrits, prostatitis etc.
Technique: Tests for the presence of protein are carried out on a clear specimen of urine
which should be filtered or centrifuged to remove any turbidity.
Boiling and Acetic Acid Test: Three quarters (3/4) of a test tube is filled with clear urine
and the upper one third (1/3) of the column is boiled for about two minutes. The development of
turbidity may be due to albumin is present. In phosphaturia turbidity will disappear on adding
dilute acetic acid.
Heller’s Test: Pour some nitric acid carefully over some urine in a test tube through a
pipette. Albumin gives a white ring at the junction which persists on heating.
Salicyl-Sulphonic (Sulphosalicylic) Acid Test: To a few ml. of urine 0.5 ml. of a 20%
aqueous solution of Salicyl-sulphonic Acid is added. Turbidity indicates the presence of protein,
but occasionally a false positive result may be due to presence of uric acid.
QUANTITATIVE TESTS FOR ALBUMIN
ESBACH’S ALBUMINOMETER: (Esbach – 1874)
It is used to estimate albumin in urine.
a) Identification: It is shaped like a test tube and is graduated in grams of dried albumin
per 100 ml of urine. Graduations in the tube range from 1 to 20.
It has a mark ‘U’ in the middle up to which filtered acidified urine is added.
It has another mark ‘R’ up to which Esbach’s reagent is added.
Esbach’s Reagent: It consists of picric acid to precipitate proteins and citric acid to
dissolve phosphates in urine. Esbach’s albuminometer is filled up to mark ‘U’ with urine.
The reagent is then poured upto the mark ‘R’. The tube is corked and stood vertically for
24 hrs. The proteins settle down and the amount of precipitate is read as g’ltr. If the urine
was diluted, the figure must be multiplied by the dilution factor.
Picric Acid – 1 gm.
*Esbach’s Reagent: Citric Acid – 2 gm
Distilled water - 100 ml.
Note: If the urine is alkaline, it is rendered acid by acetic acid. It is subsequently filtered. The
urine is then diluted to make specific gravity 1.008.
c) Advantages: It is simple and easy to perform.
It gives fairly reliable results.
It is inexpensive
d) Disadvantages: It is less precise and accurate, than currently available methods of
It is used to estimate albumin in urine.
a) Identification: It consists of a small centrifuge tube with graduation in percentage of
albumin. The graduations are in percentage from 0.02-1.70%.
It has a ‘U’ mark upto which acidic urine is filed and an ‘R’ mark up to which Aufrecht’s
reagent is filled.
Aufrecht’s Method: Fill the Aufrecht’s albuminometer with filtered urine to the mark ‘U’.
Add the Aufrecht’s reagent to the mark ‘R’. Close the tube with a rubber bung and invert
several times. Centrifuge the tube for five minutes at 2500 R.P.M. Albumin is precipitated.
Read the percentage of albumin directly from the graduations on the tube.
Aufrecht’s Reagent: Picric Acid – 1.5 G.
Citric Acid – 3.0 G.
Distilled Water to 100 mts.
Bence - Jones protein: Bence – Jones described a protein in urine which precipitates
between 50 – 600C, redissolves on boiling and precipitates again on cooling at 580C. The
presence of Bence-Jones protein is almost pathognomic of multiple myeloma. It has also
been observed in leukaemias and carcinomatous metastases in bones.
Various carbohydrates may be found in the urine. Glucose in by far the most common and
is the one of much clinical importance.
Techniques - Qualitative Test: If albumin is present in any considerable quantity, it
interferes with reduction of copper sulphate tests and should be removed by acidifying with acetic
acid or boiling and filtering.
a) Benedicts Test: To 5 ml. of Benedict’s reagent* (qualitative) add 8 drops of protein free
urine. The mixture is boiled for two minutes and allowed to cool. A yellow to red
precipitate indicates the presence of reducing sugar. This is a quite sensitive test as it
will detect 0.15 to 0.2 percent of glucose in the urine.
b) Fehling’s Test: Take 1 c.c. Fehling’s solutions No.1* and 1.2. c.c of Fehling’s solution
No.2** in a test tube and add about 3 c.c. distilled water. Boil the mixture. Then add
urine to be tested in fractions to the hot copper solution. The quantity of urine to be
added should not exceed that of the reagent. A greenish yellow to red precipitate will
appear depending upon the amount of sugar present.
A positive Benedict’s test, in most cases implies glycosuria. Benedict’s solution may
however be reduced by substances other than glucose. Homogenetistic acid (Alkaptonuria, and
excess of mucin oxalates, uric acid, creatinine or urine may reduce copper. Many drug such as
chloroform, formalin (used as preservation for urine) and ascorbic also may cause confusing
QUANTITATIVE ESTIMATION OF GLUCOSE IN URINE:
The method of choice in most laboratories is the Benedict’s Method.
Technique: Take 25 c.c. of Benedict’s Quantitative Reagent *** in small flask or porcelain
dish and add 5 – 10 grams (a good pinch) of Anhydrous Sodium Carbonate. Heat to boiling and
add the urine little by little from a burette until a chalk-white precipitate forms and the blue
colour of the reagent begins to fade. Now add urine, a drop at a time, until last trace of blue just
disappears which indicates the end point. Note the quantity of urine required to discharge the
blue colour; this contains exactly 0.5 gm. of glucose. From this percentage of glucose can be
The commonest condition of glycosuria is diabetes mellitus. But every case of glycosuria
cannot be branded as diabetes, since there are conditions like renal glycosuria in which glucose
appears in the urine due to the low threshold of the kidneys for glucose. Gycosuria is one of the
manifestations of Cushing’s syndrome, phaeochromocytoma, some liver disorders etc. Hence to
confirm the diagnosis of diabetes mellitus, tolerance test is essential.
Benedict’s Reagent (Qualitative) :
Copper sulphate (pure crystallized) - 17.3 gms
Sodium or potassium citrate - 173.0 gms
Sodium carbonate (Crystalised) - 200.0 gms
(or 100 gms of anhydrous sodium carbonate
Distilled water to make - 1000 ml.
* Fehlings Solutions No.1
Copper sulphate (Pire crystalline) - 34.64 gms
Distilled water - 500.00 ml
** Fehling’s Solutions No.2
Potassium sodium tartarate - 163 gms
Potassium hydroxide - 100 gms
Distilled water - 500 ml.
*** Benedict’s Quantitative Reagent
Copper sulphate (pure crystallized) - 18 gms
Anhydrous sodium carbonate - 100 gms
Sodium or potassium citrate AR. Or CP - 200 gms
Potassium sulfocynate AR or CP - 125 gms
Potassium ferrocyanide solution 5% - 5 ml
Distilled water to make - 100 ml
Ketone bodies are the three metabolically related compounds comprising of aceto-acetic
acid, B-hydroxybutyric acid and acetone. In certain conditions where metabolisms of fat and
carbohydrate are disturbed, the rate of ketogenesis in the liver is too great. Ketone bodies
accumulate in the blood and are excreted in the urine.
The most important condition associated with ketonuria is diabetes mellitus but ketonuria
may occur in prolonged vomiting, starvation, high-fat, low carbohydrate diet, prolonged febrile
illness, after either anesthesia and some cases of severe toxemia of pregnancy.
Rothra’s Nitroprusside Test for Acetone and Aceto Acetic Acid: About 2-5 ml of urine
is saturated with ammonium sulphate in a test tube and a few crystals of sodium nitroprusside
are added and dissolved. Now overlay with liquor ammonia. A gradually depending purple ring
because of the chemical ferro pentacyanide shows the presence of acetone. A brown or red color is
of no significance.
Ferric Chloride (Gerhart’s) Test for Acetic Acid: To about 5 ml of urine add drop by
drop a 10% solution of ferric chloride. A precipitate of ferric phosphate appears and then dissolves
in an excess of the reagent. A red bordeaux colour indicates the presence of aceto-acetic acid.
This is not sensitive test and the urine must be fresh because on standing, aceto-acetic
acid is converted into acetone which does not give a positive reaction by this test. Salicylates and
phenol when present in the urine strike a dark violet colour with this reagent.
BILIRUBINURIA (Bile pigment)
Billirubinuria is found in cases of hepatogenous and obstructive jaundice but not in pure
Iodine ring test (Smith’s): Overlay a column of urine with tincture iodine diluted with nine
times its volume of alcohol. A green ring at the zone of contact shows presence of bile pigment.
Nitric Acid (Gmelin’s) test: Soak a filter paper in urine and put a drop of commercial nitric
acid over it or pour-down the wall of test tube over a column of urine. A play colours, of which
green and violet are most distinctive indicates bile pigment.
Fouchet’s Test (Harrison’s): To 5 ml of urine 5 ml of 10% barium chloride is added and
mixture filtered. Put one drop Fouchet’s reagent on the precipitate in the filter paper.
A green or blue colour indicates the presence of Bilirubin.
Fouchet’s Reagent: Trichloracetic acid ……. 25 ml
10% Ferric Chloride ……. 10 ml
Distilled water ……. 100 ml
UROBILINURIA AND UROBILINOGENURIA
The trace of urobilinogen present in normal urine is insufficient to cause a significant positive
reaction with the tests described below:
In pathological conditions, the rate of excretion of urobilinogen may be variable and unless a
positive reaction is given by a casual sample, the following test should be carried out in a given
sample of urine voided between 2 and 4 p.m.
Schlesinger Test: To 5 ml. of urine add 3 drops of tincture iodine and then mix 5 ml. of a
10% suspension of zinc acetate in alcohol. The mixture is allowed to settle and the clear
supernatant fluid shows green florescence in transmitted light due to urobilin or urobilinogen.
Ehrlich’s Aldehyde test: Dilute Urine 1:2 with distilled water 4.5 ml of diluted urine + 0.5
ml. of Ehrlich’s Aldehyde reagent. Mix well. Run a control using undiluted urine in the similar
order. Allow it to stand for 5minutes. Observe the cherry red colour from the top down through
the test tube for positive reaction.
Ehrich’s Aldehyde reagent:
4 dimethyl aminobenzaldehyde - 4.5 g
Conc. HCI - 40 ml.
Distilled water - 160 ml.
Test for Bile Acid Excretion
Hay’s Sulphur test: Sprinkle flowers of sulphur on the surface of a column of urine in a wide
bore test tube (2 cm. diameter or more). If the sulphur granules sink immediately or on very gently
shaking the test tube, bile acids are present the urine.
HAEMOGLOBINURIA AND HAEMATURIA
Chemical tests can be used to detect the presence of red corpuscles or haemoglobin in urine,
but haematuria is best recognized by microscopy and haemoglobinuria by spectroscopy.
TECHNIQUE (Chemical test for detecting blood)
Benzidine TEST: Mix a good pinch of benzidine powder in 2ml. of glacial acetic acid in a test
tube and heat, cool it and add 5 ml. of urine. Add 2 ml. of H2O2. A green colour changing to blue
is indicative of blood or haemoglobin.
Haemoglobinuria: means presence of free haemoglobin in urine and is seen in conditions
where there is break down of R.B.C.s and liberation of free haemoglobin in blood, viz., black water
fever, kala azar, paroxysmal nocturnal haemoglobinuria, mis-matched blood transfusion etc.
Haematuira: means presence of erythrocytes in urine and is seen in the following conditions;
bleeding diathesis, drug sensitivity, subacute bacterial encdocarditis, acute glomerulonephritis,
destructive lesions of the urinary tract like carcinoma, tuberculosis and trauma.
MICROSCOPIC EXAMINATION OF URINE
Whenever proteinuria is discovered or diseases of the urinary tract are suspected, the urinary
deposits should be examined under a microscope.
Since the nature of the sediment soon changes, the urine must be examined while fresh,
preferably within 6 hours after it is voided.
Technique: Fresh urine, about 15 ml, is centrifuged for 5 -10 minutes at 1500 – 2000 r.p.m.
The supernatant fluid is decanted and sediment re-suspended in a small amount of urine (2-3
drops) remaining in the tube. A small drop of the sediment is transferred to a clean slide and
covered with a cover slip. The preparation is best examined with the condenser of the microscope
is lowered and the illumination reduced by almost closing the diaphragm. The high power is used
to identify the constituents.
The deposits may be considered under two main groups:
(1) Organized deposits and (2) Unorganized deposits
Organized Deposits: The elements which commonly form the organized deposits are red
blood cells, leukocytes (pus cells), epithelial cells, casts, bacteria and occasionally spermatozoa,
prostatic threads and foreign bodies.
In acid urine, leukocytes usually retain their normal appearance; in alkaline urine they are
swollen, granular and opaque. An occasional leukocyte may be present in normal urine, but more
than one per high power filed indicates pyuria. The causative organism may be identified by
A few epithelial cells from the bladder may be present in the normal urine, and cell from the
vulva and vagina usually contaminate a routine specimen from women. The presence of more
than a few epithelial cells in a clean or catheter urine specimen is abnormal and indicates disease
of the urinary tract.
Casts are formed by coagulation of albuminous material and cells in the renal tubules. They
rarely occur in urine which does not contain, or has not recently contained albumin and in a
general way, they have the same clinical significance as renal albuminuria.
Hyaline Casts: They are the most common variety and in small numbers may be found in
urine from healthy people. In large numbers they are found in all forms of renal disease.
Granular casts: When found in large numbers indicate tubular degeneration and occur in
nephritis (Type II nephritis) and chronic glomerulo-nephritis.
Fatty Casts: Consists of fat droplets often mixed with granular of epithelial cells.
Waxy casts: These are never found in normal urine and indicate tubular degeneration.
Casts containing organized structure:
a) Epithelial Casts: Are composed of cells from the renal tubules and show nuclei in various
stages of degeneration. Epithelial casts are usually found in acute renal disease such as
b) Blood and Leukocyte casts: These may consists of solid plugs of cells (Red or white cells)
which have become adherent to a hyaline or granular cast. They usually imply acute
disease and are seen in acute glomerulo-nephritis.
Unorganized Sediments: In general these sediments have little diagnostic or prognostic
significance. Most of them are substances normally present in solution which have
precipitated either because they are present in excessive amounts, or more frequently,
because of some alteration in the urine such as in reaction or in concentration.
Unorganized sediments consist of crystalline or amorphous material, the exact nature of
which varies according to whether the reaction of the urine is acid or alkaline.
Formation and appearance of crystals in urine depends upon pH of the urine i.e. acidic or
Crystals in acidic Urine:
These are as under:
i) Calcium oxalate
ii) Uric acid
iii) Amorphous urate
i) Calcium Oxalate
These are colourless refractile and have octahedral
envelop like structure. They can also be dumb-bell
ii) Uric Acid
They are yellow or brown rhomboid shaped seen
singly or in rosettes. They can also be in the form
of prism, plates and sheaves.
iii) Amorphous Urate
They appear as yellowish brown
granules in the form of clumps. They
dissolve on heating .When they are
made of sodium urate, they are needle
like in the form of thorn-apple. They
are passed more often in patients
They are yellowish in the form of silky
needles or sheaves.
They are passed in urine in jaundice.
They are colourless, hexagonal plates
which are highly refractile. They are
passed in urine in inborn error of
Crystals in Alkaline Urine
these are as under
i. Amorphous phosphate
ii. Triple phosphate.
iii. Calcium carbonate
iv. Ammonium biurate
i) Amorphous Phosphate
They are seen as colourless granules in
the form of clumps
or irregular aggregates. They dissolve
when urine is made acidic.
ii) Triple Phosphate
They are in the form of prisms and
sometime in fern leaf pattern. They
dissolve when urine is made acidic.
iii) Calcium Carbonate
They are in the form of granules,
spheres or rarely dumbbell-shaped.
They again dissolve in acidic urine.
iv) Ammonium Biurate
They are round or oval,yellowish
brown spheres with thorns on their
surface giving ‘thorn apple’
appearance. They dissolve on heating
the urine or by making it acidic.
They appear in yellowish sheaves,
rosettes, or rounded with radial
striations. They appear in urine after
administration of sulphonamide
EXAMINATION OF URINE
Test Observation Inference
Blue Litmus Red/No Change
Red Litmus Blue /No Change
GEORGE N PAPANICOLAOU (1883-1962)
‘FATHER OF EXFOLIATIVE CYTOLOGY’
George N Papanicolaou , American pathologist was born in Seaport town of Kymi, on May
13, 1883. George graduated from medical school in 1904 and earned his Ph D., in
Zoology 1910, and called in to military service in 1912. Dr. Papanicolaou began using
vaginal cytology of human in 1920 and continued to study for next 21 years and
developed Pap test for detection and early diagnosis of uterine cervical cancer. In 1954
he published the Atlas of Exfoliative cytology
Cytology is the study of body cells that are either exfoliated spontaneously from the
epithelial surfaces or are obtained from various body tissues and organs by different
-This is the study of cells which are spontaneously shed off from the epithelial surfaces
into body cavities or fluid.
-In this study, samples are obtained from diseased tissue by fine needle aspiration (FNA)
or aspiration biopsy cytology.
-In imprint cytology touch preparations from cut surfaces of fresh unfixed surgically
excised tissue are prepared on clean glass slides. These are fixed, stained and examined
The Papanicolaou test (also called Pap smear, Pap test, cervical smear, or smear test) is a
screening test used to detect pre-cancerous and cancerous processes in the endocervical canal
(transformation zone) of the female reproductive system.
Superficial squamous cells
Superficial Squamous cells
1) Scrapings from lateral wall of vagina and upper 1/3 rd of vaginal mucosa.
2) Estrogen have keratinizing effect (superficial squamous cells), where as progesterone
tends to arrest maturation at intermediate stage (intermediate squamous cells)
In par smear- superficial cells appear as mature polygonal cells with eosinophilic
cytoplasm and pyknotic nucleus
MATURATION INDEX-Ratio of parabasal cells, intermediate cells and superficial cells.
MI-0/40/60-estrogen phase -seen at ovulation
1) Helps in investigating case of ammenorhoea.
2) Estrogen producing tumour
3) Investigating case of infertility
Intermediate squamous cells
- In Pap smear intermediate cells appear as polygonal or boot shaped with cyanophilic
cytoplasm and have vesicular centrally placed nucleus
MATURATION INDEX-0/70/30- progesterone phase
- seen just before menstruation.
- For hormonal studies exfolative cells should be free from inflammatory cells,glandular
cells and anucleated squamous.
- In Pap smear parabasal cells appear as small round to oval cells, less mature than
superficial and intermediate cells, with cyanophilic cytoplasm, large round to oval
vesicular centrally placed nucleus.
- seen in post menopausal period, post partum period
PARASITE IN PAP SMEAR
Trichomonas vaginalis Intermediate cell
In Pap smear trichomonas vafinalis (Tv) appears as Pear shaped form with flagella along
with superficial, intermediate cells and against abundant polymorphs background.
Pap smear me also show BB shot or cannonball – aggregates of leukocytes covering the
surface of isolated, mature squamous epithelial cell which is attached to T vaginalis
Most common parasitic infection occurring in lower female genital tract.
Clinical symptoms – white discharge per vagina
DYSPLASTIC CELLS IN PAP SMEAR
Pap smear - show cells in various size and shape
Nuclei are round to oval and irregular in shape
Nuclear chromatin is coarsely granular and hyperchromatic
Increased nucleocytoplasmic (NC) ratio
MALIGNANT CELLS IN PAP SMEAR
Keratinized squamous cell Malignant cell
Pap smear – Show elongated and bizarre shaped cells and nuclei
Cytoplasm show fibrillary keratin
Nuclei are irregularly shaped and have coarsely granular hyperchromitic
EXFOLIATIVE CYTOLOGY OF BODY FLUIDS-PLEURAL FLUID
Malignant cells Mitosis
Cytoplasmic vacuoles Moulded cluster
a) Cells are seen in acini and single.
b) Cells are variable size.
c) Nuclei are irregular shaped with irregular border, hyperchromatic,
nuclear overlapping and nuclear border touches the cell border.
d) Chromatin is clumped with prominent nucleoli.
e) Cytoplasma abundant deep purple coloured.
f) Frequent mitosis.