CLINICAL PATHOLOGY

 INTRODUCTION:
 Laboratory Medicine/ clinical pathology includes study of
blood and blood elements, study of body fluids like pleural,
pericardial, peritoneal, cerebrospinal, synovial and semen,
examination of the urine and faeces(stool).
 Significance of these investigations:
 Helps in establishing a diagnosis or to confirm a
diagnosis
 Helps in treatment of the disease
 Helps in assessing the prognosis
 Helps also to rule out certain other conditions.
PRE-REQUISITES
 Proper requisition with the patient’s details are needed- like age, sex, admission

No, probable diagnosis- the test required should be mentioned clearly.

 Collection of the specimen/ sample plays an important role.

 Preservation of the specimen/ sample to avoid contamination.

 Necessary instruction to the patient for specimen collection.

 Labeling of the container should be taken care, to avoid any mix up of samples.

 Appropriate anticoagulant or a preservative to be used.

 Storage of the sample for further study if necessary

 Glassware used should be clean

 The laboratory is usually provided with the list of the

various tests performed with reference values.

PRINCIPLE
PATIENT SPECIMEN
EXAMINATION OF THE BLOOD
 This includes detailed evaluation of blood elements and hematologic parameters
which helps in assessment of the functions of blood cells like:
 Erythrocytes
 Leukocytes
 Platelets
 Erythrocytes(RBC)-biconcave cell, no nucleus, filled with hemoglobin size 7.2 µ.
Hemoglobin is the major protein in oxygen transport.
 Leukocytes:
 Granulocytes, Neutrophil, Eosinophil, Basophil, Monocytes, Lymphocytes
 These cells are involved in phagocytosis, inflammation and immunity.
 Platelets:
 Small, a nucleate cells, 2-4 µ size play a role in coagulation mechanisms.
 Hematopoiesis:
 Production of all the above mentioned cell lineages from the
haematopoietic stem cells (HSC) is called hematopoiesis. Stem cells are
located in bone marrow.
METHODS OF COLLECTION OF BLOOD
 Indications:
 Could be for 1 test or many tests related to various
departments(pathology, biochemistry and Microbiology). The
blood should preferably be collected early in the morning
before the patient has eaten.
 Sites: from where blood can be collected

 1. Capillary Blood

a).Finger tip of usually middle or ring finger

b). Ear lobe
c). Heal of sole- (done in children)
 2. Venous blood- ante cubital fossa

 3. Arterial blood
COLLECTION OF BLOOD
 1. Capillary blood:
 Skin prick Method: blood may be obtained by pricking the
skin of a finger, the ear lode or the heal of in an infant.
 Instruments:

 Needles with a flat body, having a cutting edge or a sterile

lancet or No11 bard parker blade are used.
PROCEDURE
 The site is cleaned properly with alcohol or spirit so as to kill bacteria and also
remove dirt and oils.
 later the skin is allowed to dry Next, raise the finger /heal , press the site between
ball of index finger and thumb so as to form a ridge then the lancet is plunged in to
the ridge to a depth of about 3mm and the light pressure is immediately removed.
The wound will automatically open, and the blood will start to flow freely.
 The first drop of the blood is moped off, and the next drop is used at once either
for collection in to a pipette or on to a slide, depending on the type of test needed .
2-3 blood smears may be made. Once the required blood has been collected , the
finger should be cleaned with piece of sterile cotton wool and pressure applied for
30-40 seconds over the needle prick wound.
 The capillary blood is used for the following tests:
 1. estimation of Hb% and R.B.C, W.B.C and platelets counts.
 2. preparation of peripheral smears
 3.blood grouping
 4.estimation of blood glucose levels
VENIPUNCTURE
 Amount of blood depends on the number and type of tests required. And based on
this, the proper size of syringe (2ml,5ml, or 10 ml) is used. The syringe should be
dry and sterile, if not dry, the R.B.C will lyse.
 Needles used for venepuncture are about 1- 1 ½ inches long with a medium size
bore of about 18-20 guage. The tip of the needle should be sharp.
 Procedure:
 The patient is made to sit down with the fore arm resting on a table to or lie down
with the arm resting.
 One of the veins in the ante cubital fossa is chosen which is in front of the elbow.
These veins are easily seen and do not slip aside. The upper part of the arm is
constricted using a torniquet or by placing B.P (blood pressure) cuff and inflate upto
40 mmhg.
 Then the patient is asked to clench the fist, or to open and close the fist for few
times so as to allow the veins to become prominent.
 Next identify and then feel the vein. Now clean the site with spirit or 95% alcohol
and allow the place to dry.
 Next place thumb of our left hand over the vein just below the
puncture site and apply slight traction and ‘fix’ the vein. The
syringe is taken in the right hand, and level of the needle
should face upwards, and insert the needle at an angle of 30
degrees skin. Push the needle firmly gently and teadily. Do not
be fast, and avoid going through and through to come out from
other site.
 One the needle is within the vein pull the piston of the syringe,
and the blood gets filled in to the barrel of the syringe. The
correct required amount of blood is withdrawn, and the
torniquet is removed.
 Syringe along with the needle is smoothly and gently
removed. Place a sterile guaze piece over the puncture
site and pressure is applied by flexing the arm for several
minutes, which prevents bleeding by closure of the
wound.
 The blood which is with drawn, should be immediately
transferred to the appropriate containers as per the tests
needed. This is done by first removing needle from the
syringe and the transfering blood in to different
containers
 Venous blood is necessary for the following tests:
 Estimation of E.S.R, P.C.V. etc.

 Estimation of blood constituents like sugar, urea etc

 Bacteriological and serological examinations.

 Blood grouping and cross matching.

3. ARTERIAL BLOOD:
 sometimes it may be impossible to collect blood from veins.
 In such cases arterial puncture 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
endocarditis.
CONTAINERS FOR DIFFERENT TESTS
 The containers vary, depending on the types of tests necessary
to be carried out on the venous blood obtained.
 Haematology tests:

 Usually ‘penicllin’ bottles are used, with appropriate amount of

anticoagulants used for the particular test. Now a days,
vacutainers are used, which can be directly centrifused without
transferring in to another container.
 Blood for biochemical and serological tests:

 Blood is usually collected in a dry, sterile test tube. No

anticoagulant is added, as most biochemical and serological
tests are done on the serum.
 Blood for bacteriological tests:

 Usually aseptic precautions are taken, when blood is collected

for bacteriological tests, especially, when it is done for culture.
PRECAUTIONS WHILE COLLECTING BLOOD
 All blood samples and patients should be treated as potentially
contaminated with either the virus which causes hepatitis or
AIDS a both hence strict precautions should be taken while
handling the patients and their samples.
STORAGE OF BLOOD BEFORE TESTS ARE DONE
 With in 2 hours-prothrombin time, plasma coagulation tests,
platelet count, blood smear making
 With in 2 hours- erithrocyte sedimentation rate ESR

 With in 24hrs- RBC,WBC, PCV, Haemoglobin Estimation,

Reticulocyte count.
 Blood can be stored in the refrigerator at 4 degrees C to
minimise the changes- the changes could be swelling of RBC
or didintegration of WBC, increase in osmotic fragility of
RBC, prothrombin and plasma coagulation times will be
prolonged, ESR may decrease.
 Note: blood in the container should be mixed properly for 1-2
min before being used for the tests
 If the blood is cooled ( in temperature) allow ie come back to
the room temperature ( or to 37oc) before used for the tests.
PRECAUTIONS TO PREVENT HEMOLYSIS
 All the apparatus used should be clean and dry
 Needle should not be withdrawn slowly. Avoid strong and fast
action of syringe.
 The needle should be removed from the syringe, before the
blood is flushed in to the bottles
 Blood should be mixed gently with the anticoagulant

 Do not freeze the blood. Don’t keep blood at room temperature

for long.
 ANTICOAGULANTS
 A) oxalates: these salts are unite with calcium in the
blood to form insoluble compounds and there by
deplete the blood of its calcium which is necessary for
the coagulation of blood.
 1) dried potassium oxalate: is used at a
concentration of 2mg/ml of blood.
 Disadvantages: causes shrinkage and destruction of
cells. It is often used for chemical analysis of blood.
 2) ammonium oxalate: used in case of estimation of blood
constituents. 2mg of dried salt is sufficient for 1ml of blood.
Disadvantage it causes swelling of RBC-therefore not used for
PCV, ESR or peripheral smear.
 3) double oxalate or wintrobes’ salt:

 Combination of ammonia and potassium oxalates (1.2g and 0.8

gm of these salts respectively and distilled water of 100 ml) is
used.
 Advantages: ammonia oxalates causes swelling of the cells
whereas potassium oxalate shrinks. The action of both these
salts is counter balances and thereby the cells retain their
original shape and size.
 B) CITRATES
 1. Trisodium 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 salts are relatively nontoxic and the salt is excreted by the
kidneys or utilised by the body. 3.8% solution is used as 1:4 ratio
with blood for ESR estimation (westergrn’s method)
 2. Acid citrate dextrose solution (ACD):

 Erythrocytes are better preserved in the ACD than in trisodium

citrate alone.
tri-sodium citrate 1.32g
citric Acid 0.42 g
Dextrose 1.40 g
dist. Water add to 100ml
15 ml of ACD is sufficient for 100 ml of blood.
Uses: this is used in blood transfusion
 C) HEPARIN:
 It has an affinity for blood proteins and acts as an antithrombin
and antithromboplastion. 1mg of dry heparin or 1000 I.U of
liquid heparin suffice for 10ml of blood.
 Used in haematocrit studies and blood transfusion especially
exchange and rapid transfusion as in cases of thoracic surgery,
E.S.R and osmotic fragility tests.
 D. ETHYLENE DIAMINE TETRA ACETATE (EDTA):

 Its di-sodium and di-potassium salts are used. It is the most

powerful chelating agent and thus separate the calcium ions
from the blood.
 Used for most of the haematological and chemical tests.

 It gives the best preservation of cell morphology, and is used

for cell counts, smears and platelet count.
HAEMOGLOBIN ESTIMATION
 Haemoglobin is a complex substance present in the RBC which
gives red colour to the blood. It is an iron containing protein
made up of a large protein molecule the globin, and four haem
molecules. The function of haemoglobin is to carry oxygen to
the tissues and CO2 away from it.
 Estimation of haemoglobin:

 This can be done by methods utilising any of the following

principles:
 1).colour

 2).specific gravity

 3).Iron content

 4). O2 combining capacity of blood.

 1. COLORIMETRIC METHOD:
 A). Tallquist method:
 Compares the colour of drop of blood drying on a piece of
blotting paper to reference standards of coloured paper in a
booklet. This is very quick and easy but is inaccurate.
 B). Alkaline haematin method:

 Here hb are converted to alkaline haematin by addition of N/10

Naoh. Here carboxy haemoglobin, methhaemoglobin and
sulphahaemoglobin are converted but not foetal Hb hence this
method is used to detect the presence of presence of foetal Hb.
 C). Haldane method:

 Here Hb is exposed to CO so carboxy Hb is formed. This test is

accurate but CO is dangerous and toxic, so not preferred in labs.
 D) haldane method: here Hb is exposed to CO so carboxy Hb is formed.
This test is accurate but CO is dangerous and toxic, so not preferred in
labs.
 E) dare method:

 Undiluted blood is spread in thick films between glass discs and seen for
direct matching.
 (f). Spencer method:

 Here colour of diluted oxy Hb is matched visually.

 G) photo electric method:

 Cyanmethhaemoglobin method:

 The most perfect and accurate method. It is internationally recommended

for best quality control but this is not done as it is time consuming. It’s a
long procedure and all apparatus should be available. Hb is 1 st converted to
meth hb and then to cyanmeth Hb by the additions of Na or k cyanide
potassium ferricyanide. This solution is called as Drabk in solution. It is
highly poisonous and should not be pipetted by mouth. However this
method is widely used in most private labs.
 Procedure: 5ml of Drabkin solution is pipetted in to cuvette to
which 0.02ml ml of blood is added. Optical density is read &
value is recorded.
 Specific gravity methods:

 A drop of blood is allowed to fall in to solutions of cuso4 of

varying specific gravities and marking whether the drop rises
to surface or sinks in to the bottom. If the blood drop sinks, its
specific gravity is grater than that of cuso4 solutions. This is
mostly used for screening of blood donors.
iii). Chemical method: where iron content is the criteria, it is a
complex, difficult and time consuming method but is accurate.
iv) Gasometric method:
Based on O2 combining capacity of blood. It measures only
functional hb. It is difficult and time consuming. It is used as
another ‘reference method’ but not used for routine lab use.
ESTIMATION OF HAEMOGLOBIN
SAHLI’S METHOD:
AIM: to estimate hb in blood by acid haematin method.
Apparatus: sahli’s haemoglobinometer tube and apparatus,
pippete, glass rods, N/10 HCL, blood, distilled water, spirit,
sterile cotton wool and pricking needle.
Method:
Take N/10 HCL upto lowest mark on the percentage scale of
sahli’s tube. Prick finger under sterile conditions and draw
blood in to the graduated tube immediately by repeated filling
and emptying of the pipette till it is completely free of blood.
Mix it with N/10 HCL already present, with stirrer and wait for
10 mts. Acid haematin is formed and shows brown colour. Add
distilled water till the colour approaches that of the standards.
 Take care in adding the last few drops. Take the reading on the
graduated tube as gm/100ml of blood.
 The colour matching should be against natural light and
reading noted at the lower meniscus.
 Normal values:

 males-13-16ms% females 12 to 15gm%

 Variation of Hb concentration:

 i)increased

 Physiological: new borns, after muscular exercises, emotional

states and at higher altitudes.
 Patholoogical: polycythaemia , cyanotic heart diseases and
burns
 Ii) increased: in anaemias especially iron deficiency anaemia ,
leukemia.
 Precautions:
 Wait for atleast 10 min for formation of acid haematin

 Remove stirrer while adding distilled water and also

while taking the reading
 Tap water can not be used as the ph varies and accurate
values may not be obtained.
HAEMATOCRIT OR PACKED CELL
VOLUME(PCV)
 Haematocrit is the volume of red cells expressed as a
percentage of volume of whole blood in the sample. The
venous pcv is almost the same as that obtained from a skin
puncture. Anticoagulant used are EDTA, double oxalate or
dried heparin.
 Methods:
 1). Using Wintrobe’s tube
2).using capillary tubes (micro haematocrit method)
WINTROBE’S TUBE
 Procedure:
 Fill the tube with anticoagulated blood sample till mark 100 on top with help of
pasteur’s pipette or a syringe with a long needle.
 To avoid false reading, filling should be without any air bubbles in the blood
column.
 Centrifuge for ½ hr at 3000rpm. Blood is now separated into 3 layers- a column of
RBC at the bottom, middle layer of WBC and platelets and the top most fluid
column formed by the plasma.
 Height of the column of blood occupied by the packed RBC gives the PCV in
percentage. Roughly, the value is 3 times the haemoglobin value, and RBC count is
1/3 rd Hb value (in millions) eg for 15 gm% Hb: PCV=45 vol% RBC Count =5
millions
BUFFY COAT
1mm of this buffy coat rouhly corresponds to total leucocyte
count of 10,000. if less than 1mm it is leucopenia and if more
than 1mm- leukocytosis. Platelets form a thin layer over WBC.
This is pinkish to white in colour and has no clinical
significance.
Plasma colour varies in different conditions:
plasma - pale yellow or straw coloured
yellow - in jaundice
pink - in haemolysis
creamy white - in hyper lipidemia
brown coloured - in case of methhaemoglobinaemia.
MICRO HAEMATOCRIT OR CAPILLARY
TUBE METHOD

 It is commonly used in well equipped labs . Capillary tubes

coated with anticoagulants are filled with blood.

One end of the tubes are sealed with wax and kept in centrifuge
for 3 minutes at high speed. By reading packed cell height
and height of the entire sample, the haematocrit is calculated.
On an instrument given along with the special centrifuge.
 USES OF BUFFY COAT:
 Leukemia
 L E cells

 Detection of plasma cells

 Haemoparasites

 SOURCES OF ERROR:

 In adequate mixing of blood

 Irregularity of bore of tube(broken)

 Incomplete packing or centrifuging

 Normal values:

 Adult males – 40 to 55%

 Females – 37 to 47%

 Children – 35 to 37%
BLOOD CELL COUNTS
 Haemocytometers:
 It is instrument used for counting blood cells. This box
contains a counting chamber and two pippettes. One for RBC
and other for WBC. The counting chamber most commonly
used is Ivy’s chamber with an improved neubauer ruling.
 It has two ruled stages separated by a small gutter.

 The two stages are in turn separated from the two ridges – one
on each side by the gutter. The two stages are in turn separated
from the two ridges- one on each side by the gutter. The
surface of the ruling is 1/10mm above the surface of stage.
 It is a large ruled area of 9 mm2, divided into 9 squares. The
IMPROVED NEUBAUER RULING:


corner squares are divided in to 16 smaller squares. These 4
corner squares are used for counting WBC.
 The centre square (is divided into 25, fine squeres- the four
corner and one central ) are used for counting RBC. Out of
the chambers, double ruled chambers are better for counting.
Spencer bright line chamber is the best but is expensive.
 Neubauer’s chamber is a thick glass plate with the size of a
glass slide (30x70x4mm). The counting region consists of two
square shaped ruled areas. There are depressions or the moats
on either side or in between the areas on which the squares are
marked thus giving an “H” shape.
 The ruled area is 3mm2 divided into 9 large
squares each with a 1 mm2 area. The large central
square (which can be seen in its entirely with the 10X
objective), is divided into 25 medium squares with
double or triple lines. Each of these 25 squares are is
again divided into 16 small squares with single lines,
so that each of the smallest squares has an area of
1/400 mm2.
PROCEDURE FOR CHARGING

 The counting chamber and the cover slip should be clean and
place it carefully on the microscope stage. Mix and roll the
pippette well, discard the first few drops and placing the tip of
the pipette at the edge of the coverslip, allow a small volume of
fluid to flow down in between the chamber, and coverslip. The
drop is attracted under the coverslip by capillary action.
 Uses of Neubauer chamber and number of squares to be
counted:
 1. RBC count – central square- 5 big squares in centers with small squares.
 WBC count- corner square (4)
 Eosinophil count- all central and corner squares (9)
 Cell counts of cerebrospinal fluid and other body fluids. – all central and
corner squares (9)
 Semen analysis- count – corner squares(4)
 Platelet count- 25 bit squares in centers with 16 small squares each.
TOTAL WBC COUNT
 APPARATUS:
 Haemocytometer with WBC pipette, sterile needle, cotton
swab, spirit and thomas diluting fluid, and blood sample. The
WBC pipette is smaller and has a white bead. Markings are
0.5, and 11. compared to RBC, as wbc are lesser in number,
dilution is also less. Procedure take blood upto 0.5 mark, fill
dilution fluid up to 11 mark, hence dilution factor is 1/20. cells
in the 4 corner squares each 1mm2 are counted.
 Thomas fluid is the W.B.C diluting fluid. Its composition is as
follows:
 Glacial acetic acid- 1.5 ml (to lyse RBC) 1% gentian violet in
water – 1ml ( to stain the nucleus- mixed in 98 ml of distilled
water.
PRECAUTIONS
 Precautions:
 Allow cells in the chamber to settle for several minutes.

 Counting done with condenser diaphragm of microscope partially

closed to make the cells stand out clearly.
 Errors: could be due to varies causes

 1. due to nature of sample -coagulated blood

 2. operator’s errors - faulty technique

 3. errors due to equipment - inaccuracies in graduation

 Formula:

 Total count of leucocytes is calculated

 Total no of cells = no of cells counted × dilution factor× depth factor
 chamber area counted
 Tatal counts normal range:
 Adults- 4,000-10,000/ cu mm
 at birth 10,000-25,000/ cu mm
 1-3 years 6,000- 18,000/ cumm
 4-7 years 6,000 – 15,000 / cumm
 8-12 years 4,500 -13, 5000/ cu.mm
 Leucocytosis incresed WBC count

 Muscular exercise

 Sever haemorrhage

 Infections

 High temperature

 ( leucopenia) decreases WBC count:

 Aneamia

 Typhoid & viral fevers

 Bone marrow depression

TOTAL RBC COUNT

 Apparatus:
 Haemocytometer, RBC pipette, sterile needle, cotton swab,
spirit, hayem’s dilution fluid, and sample of blood
 Filling the pipette:

 the pipette must be clean and dry. Suck the blood to mark 0.5
holding the same almost horizontalloy. Should the blood go
slightly beyond the mark draw it back by touching the tip of
the pipette to a moistened towel.
 Quickly wipe off the blood adhering to the tip, plunge it into
the diluting fluid and suck the fluid upto the mark 101. slightly
rotating the pipette meanwhile. Close the ends of the pipette
with the fingers, and shake gently for about a minute or two as
it gets mixed with blood.
 Counting the erythrocytes:
 Allow at least 3-5minutes for the red cells to settle. The R.B.C
must be evenly distributed over the whole area. Count the
erythrocytes in 16 small of the RBC area (I,e total 5 square &
each has smallest squares) squares under the high power
objective. Count the cells which touch the lower and left sides
and omit those touching the upper and right sides in order to
avoid recounting.
 Calculation:

Total no of cells = no of cells counted × dilution factor× depth factor

chamber area counted
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

 Normal values:

 R.B.C count adult male 4.5 – 6.0 millions/ µ

 female – 4.0-5.5 millions/ µ

 Increased RBC level:
 Age at birth more

 Cases of haemoconcentration

 Central cyanotic states

 Polycythemia

 Decreased RBC level

 Old age

 Anaemias types

 Composition of hayem’s fluid:

 Mercuric chloride= 0.5 gm

 Sodium chloride=1 gm

 Sodium sulphate = 5 gm

 Distilled water =200 ml

PLATELET COUNT
 Platelets are small, colourless withy a diameter of 2 to 4
microns. They help in coagulation, integrity of vessel wall &
for clot retraction.
 Procedures:

 1. Indirect method:

 Done with peripheral smear & then proportion of platelets is

composed of no of RBC.
 2. Direct method:

 Capillary or venous blood is used. Anticoagulant of choice is

EDTA as it reduces clumping of platelets. Formalcitrate is used
as diluting fluid.
 Method:
 0.02 ml of blood is mixed with 2ml diluting fluid for 2 min &
then charge the improved Neubauer chamber. Place this
chamber in a petri dish placed on a wet filter paper & leave
for 10 min. this is for the platelets to settle on the surface of
the chamber. Then using high power lens with lowered
condenser, platelets are counted. The counting is done in RBC
fields of 1mm2 area. The total value is calculated from given
formula.
CALCULATION
 Area counted = 1 mm2: dilution: 17100
 Standard depth = 1/10 mm

 Number of platelets(x) = n×100x 10 = nx 1000/ mm

 Normal count 1-3 lakhs/ cu.mm

 Variations:

 (a)increased: ( thrombocytosis)

 1. chronic myeloid leukemia

 2. polycythaemia vera

 3. post splenectomy cases.

 (b). decreased: (thrombocytopenia)

 1. idiopathic thrombocytopenia

 2. Aplastic anaemias

 3. megaloblastic anaemias
COAGULATION TIME
 The following points highlight the top two methods for clotting
time. The methods are: 1. Capillary Tube Method 2. Lee and White
Method.
 1. Capillary Tube Method:

 Procedure:

 i. Clean the tip of a finger with spirit. ii. Puncture it upto 3 mm

deep with a disposable needle.
 iii. Start the stopwatch.

 iv. Fill two capillary tube with free flowing blood form the puncture
after wiping the first drip of blood.
 v. Keep these tubes at body temperature. vi. After 2 minutes, start
breaking the capillary tube at 1 cm distance to see whether a thin
fibrin stand is formed between the two broken ends.
 vii. Stop the watch and calculate the time from average of the tow
capillary tubes.
 Disadvantages:
 (i) Method is insensitive.

 (ii) Method is unreliable.

 Advantages:

 It can be performed when venous blood cannot be

obtained.
 Normal clotting time is 4 to 7minutes.

 ulate the time from average of the tow capillary tubes.

 2. Lee and White Method:
 Procedure:
 i. After cleaning the forearm, make a venepuncture an draw 3 ml of blood
in a silicon-sided glass or plast syringe.
 ii. Start the stopwatch.
 iii. Transfer 1 ml of blood each into 3 glass tubes which at kept 37° C in a
water bath
 iv. After 3 minutes tilt the tubes one by one every 30 second.
 v. The clotting time is taken when the tubes can be title without spilling of
their contents.
 vi. Calculate the clotting time by average of 3 tube.
 Normal values: 5 to 11 min ( 6 to 9 minutes usually)
 Advantages:
 (i) More accurate and standard method.
 (ii) Test can be run with control.
 Disadvantages:
 (i) The temperature should be maintained because higher
temperature accelerates clotting.
 (ii) The diameter of the glass tubes should be uniform
because clotting is accelerated in narrow tubes.
 (iii) Vigorous agitation of the tubes should be avoided as
it shortens the clotting time.
CLINICAL APPLICATION OF
CLOTTING TIME:
CLOTTING TIME IS
PROLONGED IN
FOLLOWING CONDITIONS:
(I) SEVER DEFICIENCY OF
COAGULATION FACTORS.
(II) AFIBRINOGENAEMIA.
(III) ADMINISTRATION OF
HEPARIN.
(IV) DISSEMINATED
INTRAVASCULAR
COAGULATION (DIC).
(V) ADMINISTRATION OF
DRUGS SUCH AS
ANTICOAGULANTS.
BLEEDING TIME
 Bleeding time is duration of bleeding form a standard
puncture wound on the skin which is a measure of the
function of the platelets as well as integrity of the vessel
wall. This is one of the most important preliminary
indicators for detection of bleeding disorders. This is also
the most commonly done preparative investigation in
patients scheduled for surgery.
 A small puncture is made on the skin and the time for
which it bleeds is noted. Bleeding stops when platelet plug
forms and breach in the vessel wall has scaled.
 Methods for Bleeding Time:
 1. Finger tip method

 ADVERTISEMENTS:

 2. Duke’s method

 3. Ivy’s method

 1. Finger Tip Method:

 Procedure:

 ADVERTISEMENTS:

 i. Clean the tip of a finger with spirit.

 ii. Prick with a disposable needle or lancet.

 iii. Start the stop-watch immediately.

 iv. Start gently touching the pricked finger with filter paper till blood spots continue to
be made on the filter paper.
 v. Stop the watch when no more blood spot comes on the filter paper and note the time.

 Disadvantages:

 (i) It is crude method

 (ii) Bleeding time is low by this method

 Normal bleeding time 1-3 minutes.

 2. Duke’s Method:
 ADVERTISEMENTS:

 Procedure:

 i. Clean the lobe of a ear with a spirit swab.

 ii. Using a disposable lancet/needle, puncture the lower edge of the earlobe to
a depth of approximately 3 min.
 iii. Start the stop-watch immediately.

 iv. Allow the drops of blood to fall on a filter paper without touching the
earlobe and then slowly touching the blood drop gently on a new area on the
filter paper.
 v. Stop the watch when no more blood comes over the filter paper and note the
time.
 Advantages of the Methods:

 (i) The ear lobule has abundant subcutaneous tissue and it vascular.

 (ii) Flow of blood is quite good.

 Normal Bleeding Time 3-5 Minutes:

 IVY method:
 The IVY method is the traditional format for this test. While
both the IVY and the Duke method require the use of a
sphygmomanometer, or blood pressure cuff, the IVY method
is more invasive than the Duke method, utilizing an incision
on the ventral side of the forearm, whereas the Duke method
involves puncture with a lancet or special needle. In the IVY
method, the blood pressure cuff is placed on the upper arm
and inflated to 40 mmHg. A lancet or scalpel blade is used to
make a shallow incision that is 1 millimeter deep on the
underside of the forearm.
CONT…
 A standard-sized incision is made around 10 mm long
and 1 mm deep. The time from when the incision is
made until all bleeding has stopped is measured and is
called the bleeding time. Every 30 seconds, filter paper
or a paper towel is used to draw off the blood.
 The test is finished when bleeding has stopped.

 A prolonged bleeding time may be a result from

decreased number of thrombocytes or impaired blood
vessels. However, the depth of the puncture or incision
may be the source of error.
 Normal values fall between 3 – 10 minutes depending on
the method used.
PROTHROMBIN TIME (QUICK’S METHOD)
 Definition:
 It is defined as the time required for clotting of citrate or
oxalate plasma in a glass tube after the addition of calcium
and tissue thromboplastin.
 Prothrombin is transformed into thrombin by a clotting
factor known as factor X or prothrombinase; thrombin then
acts to transform fibrinogen, also present in plasma, into
fibrin, which, in combination with platelets from the blood,
forms a clot (a process called coagulation).
 Procedure:
 1. Take 4.5 ml of patients blood in to a tube containing
0.5ml of 0.1M sodium oxalate or 0.5 ml of 3.8% sodium
citrate. Mix blood well, and centrifuge at 2,000 -3,000
rpm/10min. Remove the plasma.
 2. make a control plasma in the same way.

 3. place 0.1ml of saline thromboplastin suspention and

0.1 ml of 0.15 m cacl2 in to 10 x 75mm test tube and
agitated slightly and they are placed in water bath at
37oC
 After 1 min 0.1 ml of the plasma (kept at 37oC) is added to
the above cacl2 thromboplastin mixture and start stop watch.
After 5-7 sec, gently mix them, and the time is recorded.
 The procedure is twice, and average time is noted
 Normal value: 14 ±2seconds.
 Abnormal values: seen in coagulation disorders, especially
stage 2 defect in the coagulation mechanism.
SEROLOGICAL AND IMMUNOLOGICAL TESTS AND
OTHER BLOOD TESTS
 Serology is the scientific study of blood serum. In
practice, the term usually refers to the diagnostic
identification of antibodies in the serum. Such antibodies
are typically formed in response to an infection, against
other foreign proteins or to one’s own proteins( in
instances of autoimmune disease).
 Serological tests may be used for :

 Early diagnosis of infection such as viral hepatitis and

typhoid.
 Detection of infections in which it is difficult to isolate
organism. Eg: syphillis
CONT…
 In forensic medicine, generally to link a perpetrator to a
piece of evidence.
 There are several serology techniques that can be used
depending upon the antibodies being studied. These
include
 Agglutination

 Precipitation

 Enzyme – linked immunosorbent assay (ELISA).

 Complement faxation test,

 Immunofluorescence etc..
 AGGLUTINATION TEST
 Agglutination is clumping together of cells having
surface antigen with its specific antibody. Visible clumps
thus formed are known as agglutinates as antigen-
antibody complexes formed are insoluble. These are
commonly used for detection of antibodies in serum such
as widal test for typhod. Agglutination is of usually two
types:
 1. slide agglutination

 2. tube agglutination
 PRECIPITATION TEST
 Antigen and antibody are in soluble form. They react to
form insoluble visible precipitate.
 Well felix test for rickettsial diseases is an example of
precipitation test,
 vinereal disease research laboratory (VDRL) test for
syphillis is a floccullation test, a modified form of
precipitation test.
 ELISA TEST
 The enzyme linked immunosorbent assay (ELISA) is a
biochemical technique used to detect the presence of an
antibody or an antigen in a sample. Performing an ELISA
involves at least one antibody with specificity for a particular
antigen.
 The antigen antibody complex, which are immobilized on
solid support, is detected by detection antibody labelled with
an enzyme. Addition of substrate leads to color development
due to enzyme action.
 Types:
 1. Direct ELISA:
 it is used for detection of antigen. The wells of microtiter
plate are coated with antibody specific to antigen being
tested, eg detection of hepatitis B surface antigen.
 2. Indirect ELISA: it is used for detection of antibodies.
Antigen is coated on support and antibodies present in
sample bind to it,
 eg: detection of anti- HIV antibodies.

 Enzymes commonly used for labeling are horseradish

peroxidase (HRP) And Alkaline Phosphatase(ALP). The
intensity of the color produced is proportional to the amount
of attached enzyme and therefore to the amount of unknown
antibody/ antigen in the patient’s serum.
 HEPATITIS B SURFACE ANTIGEN
(HBSAG, AUSTRALIA ANTIGEN)
1. The detection of HBsAg in serum is the most important
diagnostic parameter for a viral hepatitis B virus is
transmitted through infected body fluids. Acute infection
causes hepatitis presenting with jaundice and vomiting, while
chronic infection can lead to liver cirrhosis and
hepatocellular carcinoma.
2. This antigen can be detected in the serum 2 or 3 weeks
before onset of the disease features and attains a maximum
titer on development of characteristic symptoms (icterus,
elevated GOT).
3. The special significance of detection of HBsAg is in the
screening of blood donors.
 VDRL TEST
 Veneral disease research laboratory(VDRL) is a blood
test for syphillis and is named after venereal disease
research laboratory where it was developed.
 Principle:

 A phospholipid (cardiolipin), derived from beef heart

muscle together with cholesterol and lecithin is used as
an antigen. Antibodies produced by a patient with
syphillis reacts with this antigen, leading to foaming of
test tube fluid, known as flocculation. These floccules
can be observed with naked eye, hand lens or under a
low power objective of a microscope when the reaction
is weak.
 WIDAL TEST

 The widal test is a serological test for typhoid fever. This

test demonstrates the presence of somatic (O) and
flagellar (H) agglutinins to salmonella typhi in the
patients serum the recommended method of performing
the widal test is by the tube agglutination technique.but it
can be performed by the slide method also.
 TUBERCULIN SKIN TEST
 Tuberculin skin test is a screening tool for tuberculosis(TB).
Mantoux test, also known as purified protein derivative
(PPD) test is the most common form of this test perfoemed.
 Procedure:

 1. above 0.2ml ppd is injected intradermally and read 48-72

hours later. A person who has been exposed to the bacteria is
expected to mount an immune response in the skin
containing the bactetial proteins.
 2. the reaction is read by measuring the diameter of
induration (palpable raised, hardened area) across the
forearm in millimeters. Erythema (redness) should not be
measured.
 3. the induration of more than 10mm indicates positive tuberculin
test and less than 10mm, bvut more than 5mm considered doubtful
and test must be repeated. However, in HIV/AIDS or in
immunocompromised patients even indutation of more than 5mm is
considered as positive test.
 A tuberculin skin test is done in “

 People who have been in close contact with someone known to have
TB.
 Health care workers who are likely to be exposed to TB.

 People with TB symptoms, such as an ongoing cough, night sweats

and weight loss for no other reason.
 People who have had an abvnormal chest x-ray.

 Should no do:

 Known TB infection

 Positive tuberculin skin test in the past.

 Skin rash that would make it hard to read the skin test.
 BONE MARROW EXAMINATION
 Introduction:
 Bones are composed of cortex and medulla. Medulla is
made up of cancellous bones, along with bone marrow,
which can be either red or yellow marrow. Red marrow
contains hemopoietic cells where as yellow marrow is
made up of fat tissue.
 Bone marrow is an

important indispensable
adjunct to the
study of diseases of blood.
 INDICATIONS FOR B.M STUDY:

 1. Red cell disorders.eg: megaloblastic anaemia,

 2. white blood cell disorder eg: leukemias

 3. platelet disorders.eg: idiopathic thrombocytopenic

purpura
 4. storage disorders eg: gaucher’s disease

 5. parasitic disorder. Eg: kala azar

 6. plasma cell disorders eg: multiple myeloma

 7. metastatic deposits- for any metastatic deposits. Eg:

ca. breast, ca. prostate
 8. staging purpose: for staging of lymphoma

 9. myeloproliferative disorder. Eg: chronic myeloid

leukaemia.
 TYPE OF BONE MARROW STUDY
 There are 3 ways in which marrow can be obtained.
 1. needle aspiration

 2. trephine biopsy

 3. surgical biopsy

 Needle aspiration:

 Is a simple, safe relatively painless method& can be

repeated many times. Can be done on an ‘ out patient’
basis. It is done with salah’s or klima’s needle
 Trephine biopsy
 Is a less simple but widely used method. Is superior to
needle aspiration. In certain circumstances like bone
marrow involvement as in lymphomas or non-
hematological malignancy like prostate cancer, GIT cancer.
it provides a larger piece of marrow. it is done with
jamshidi’s or islam’s needle.
 Surgical Biopsy:

 Done in operation theatre in cases of repeated dry tap. Gives

larger piece of marrow. It is dangerous and need to be
cautious with bleeding disorders, leukemias etc.
 Aspiration:

 Trephine biopsy may be combined by using a spherical

small bore needle called jamshidi’s needle.
NEEDLE ASPIRATION
 1. individual cells are well preserved. After staining the
smears, the differences of one cell from the other can be
recognised to a far greater degree.
 2. use for cytogenetics like in Chronic myeloid
leukemia (CML) for culture.
 Disadvantages:

 1. arrangement of cells as in the marrow & relationship

of one cell to the other is more or less destroyed.
 SITE FOR BONE MARROW ASPIRATION
 1. adults – sternum, iliac crest, anterior & posterior iliac
spines, spinous processes of lumbar vertebrae.
 2. children- iliac crest, medical part of proximal tibia.

 MARROW ASPIRATION:

 Sternal site:

 Above or below manubrium against 2nd (intercostal

space) in mid-line.
 Apparatus B.M :
 Aspiration needle, 2 petri-dishes with EDTA, syringes
2ml & 20ml, 2% xylocaine, cotton& spirit.
 PROCEDURE:

 Patient in made to lie flat on his back. Skin over the upper
part of the sternum is cleansed with spirit.
 In a male patient, the site should be shaved 1-2ml of 2%
xylociane is taken in a sytinge & skin. S.C tissue, &
periosteum are infiltrated.
 Wait for 2-5 minutes. Take the aspiration needle.
PETRIDISH
 First adjust the screw approximately by seeing the
xylociane injection needle through the skin
vertically down to the bone in a drilling mechanism
& feel for the “Give in ” sensation.
 Once in the marrow remove the stylet &
immediately fit it with a 20 ml syringe& aspirate the
marow up to 0.1-0.2ml. Patient experiences suction
pain- one sign to know needle is in the marrow.
 Once aspirated remove the needle with one go &
apply ‘pressure’ with cotton over the site for 2 to 5
minutes.
 Meanwhile transfer the aspirated material into the 2
petridishes with anticoagulant.
 Bone marrow particles are seen as tiny grey-white bits at the
bottom.
 Take some of the bits on to a slide& make a smear similar to
peripheral smear but a little extra pressure is used to gently
press the particles &drag them. Allow the slides to air dry.
 Some of the smears made are put in to glass jars which contain
fixative-ethanol for the purpose of special stains like PAS
(periodic acid stain).
 Then stain for routine purpose, stain with leishman’s stain.
The preparation is satisfactory only when marrow particles as
well as free marrow cells are identifies in the film.
 TYPES OF SMEAR
 1. routine smear
 2. touch smear

 Examination of smear:

 This is done first under low power. Look for cellularity-

megakaryocytes, granulomas, clumps of tumor cells.
Then count under oil immersions. A differential count of
bone marrow is called myelogram % of myeloid series.
SERIES OF ERYTHROBLASTS
 DIFFERENT CELL COUNT OF MARROW IN AN ADULT

type of cell % cells

myeloblast 0-3
promyelocyte 3-10
neutrophilic 4-10
meta myelocyte 3-8
lymphocyte 5-10
monocyte 1-3
plasma cell 1-5
pro erythroblast 0-1
early normoblast 1-5
intermediate normoblast 5-20
late normoblast 5-15
mega karyo cyte 0-2
macrophage 0-1
 1.cellularity:
 40%fat cells, 60% marrow cells- normocellular in a normal
adult. Can be normol , hypo or hypercellular.
 2. mycloid and erythroid ratio: (Normal)-2:1 to 4 :1
 This is done by counting at least 500cells and expressing the
cells as ratio of myeloid and erythroid cells.
 3.Erythropoiesis:
 Marrow response: normal/ increased/ decreased
 Maturation : normoblastic/ megaloblastic
 4.Myelopoisis:
 It is examined for
 Normal response: normal/ inceased/ decreased
 5.Maturation arrest
 mor[phological abnormalities: hyper granular/ absent granules
 Abnormal cells
 Megakaryopoisis:
 They are examined for
 Number
 Morphology
 Lymphocytes:
 Normal/incresed/ abnormalplasma cells: normal/
increased
 Others: metastatic tumor cells, parasites, fungus or any
other abnormal cells.
 Iron stores:
 Iron stores are evaluated by prussian blue reaction (perl’s
stain). Iron stores are commented as normal/ inceased/
decreases or nil.
 DRY TAP
 In certain conditions, bone marrow cannot be aspirated
in to the needle or syringe when attempted. Then it is
called dfry tap.
 Eg: aplastic anaemia, myelofibrosis, when marrow is
tightly packed with leukaemia cells.