CLINICAL TRAY REVISION

PRACTICAL
BATCH : B
 Anticoagulants
• These are the chemical substances that prevent coagulation of
blood.
• There are many anticoagulants used for different indications.
• They may be used in bulbs or vacutainers.
Anticoagulant Bulbs
EDTA (Ethylene Diamine
Tetraacetic Acid):
• Dipotassium salt of
EDTA is used.
• Used as 1-2mg per ml of
blood
• Mechanism : Acts as a chelating agent and
binds calcium in unionized form
• Uses:
– Routine hematological investigations (Hb
estimation, WBC count, RBC count, platelet
count)
– Anticoagulant of choice for platelet count
• Disadvantages:
– Not good for coagulation studies
 Double oxalate bulb:
=
• Contains ammonium
=%÷ᵈ
oxalate (6 parts) and
Potassium oxalate (4
parts). It is used in
, I
concentration of 2mg
for 1ml of blood.
• The combination
minimizes
morphological changes
in the erythrocytes
• Mechanism: Precipitates calcium as insoluble calcium
oxalate
• Uses:1)For routine hematological examinations
2)ESR by Wintrobe’s method
• Disadvantages:
– Vacuolation and other changes in leucocytes
– Phagocytosis of oxalate crystals by the cells
– Cannot be used for the determination of blood
urea, nitrogen and potassium
Sodium citrate bulb:
• Contains trisodium
citrate (3.8% w/v)

weight /V01
• Mechanism: Calcium is bound in unionized
form.
• Uses:
– Coagulation studies (used in ratio of 1:9)
– ESR by Westergren’s method (used in ratio of 1:4)
– Used as CPD (Citrate Phosphate Dextrose) and
ACD (Acid Citrate Dextrose) for blood transfusion
purposes
Heparin:
• Heparin sodium is
supplied in vials in
various strengths.
• 0.1 to 0.2 mg per ml of
blood
• Mechanism: Neutralizes thrombin and inhibits
coagulation at various steps
• Uses:
– Coagulation studies, LFT, RFT, estimation of electrolytes
– Osmotic fragility test
– Blood transfusion purposes especially in case of paediatric
patients who require less than 50 cc of blood
• Disadvantages:
– Imparts a faint blue colouration to the films stained by
Romanowsky dyes
– Causes clumping of leukocytes not to be used for cell
counts
– Blood for transfusion purposes cannot be stored for more
than 24 hours
• Plain Bulb:
– No anticoagulant used
– Use: Serological tests
(e.g. Thyroid function
test, Widal test, HIV,
HBsAg, HCV, Drug
levels),
Immunohematological
tests (e.g. blood
grouping)
 Difference between plasma and serum
Plasma Serum
Fluid obtained when anticoagulated blood has been Fluid obtained when coagulated blood has been
centrifuged centrifuged

Anticoagulants are needed for separation Anticoagulants are not needed

Fibrinogen is present in plasma Fibrinogen is absent
Do not need standing, it could be centrifuged as soon as Serum takes a longer time to prepare
it’s been mixed thoroughly

Transfusion is done for trauma patients, patients with Used for enzyme tests and hormone tests
severe liver diseases
 Vacutainers
• Sterile glass tube with
coloured rubber stopper
vacuum seal inside
facilitating the drawing
of a predetermined
volume of liquid.
• Different colour
indicates different
additives.
 Tube cap colour Additive used Amount of blood drawn

Purple or lavender Potassium EDTA 2-3ml

Red No additive 3-5 ml
Green Sodium heparin or lithium 3 ml
heparin

Light blue Sodium citrate 2-3 ml

Grey Sodium fluoride and oxalate 2 ml
Syringe and Needle
• A syringe is a simple
reciprocating pump consisting of
a plunger that fits tightly within a
cylindrical tube called a barrel.
• The barrel of a syringe is made
of plastic or glass, usually has
graduated marks indicating the
volume of fluid in the syringe.
Parts of a syringe:
• A disposable hypodermic
needle is a very thin,
hollow metallic tube with
a sharp tip that contains a
small opening at the
pointed end.
• The main system to measure the diameter of the
needle is Birmingham gauge method.
• Needles in common medical use range from 7
gauge (the largest) to 33 gauge (the smallest).
• 21-gauge needles are most commonly used for
drawing blood for testing purposes.
• 16- or 17-gauge needles are most commonly used
for blood donation.
• Uses:
– administer injections
– infuse intravenous therapy into the bloodstream
– collection of blood
– FNAC (Fine needle aspiration cytology)
 Hemoglobinometer
• A hemoglobinometer is a medical measuring device
of hemoglobin blood concentration.
• Indications of hemoglobin estimation:
– To determine presence and severity of anemia
– Screening for polycythemia
– To assess response to specific therapy in anemia
– Estimation of red cell indices
– Selection of blood donors
 • Different methods of hemoglobin estimation:
– Colorimetric methods:
1. Visual colorimeter
2. Photoelectric colorimeter
– Gasometric methods
– Specific gravity methods
 Sahli’s Hemoglobinometer
• Principle: It is a visual/colour comparison method.
Blood is mixed with an acid solution so that hemoglobin is
converted to brown-colored acid hematin.
This is then diluted with water till the brown colour matches
that of the brown glass standard.
The hemoglobin value is then read directly from the scale.
 • Parts of the
hemoglobinometer:
1. Sahli’s graduated
hemoglobin tube (marked in
grams percent g% (2-24) and
percentage % ( 10 -140)
2. Sahli’s pipette or
hemoglobin pipette (marked
at 20µl or 0.02 ml). No bulb
3. Comparator with a brown
glass standard. Opaque white
glass is present at the back to
provide uniform illumination.
4. Stirrer: Thin glass rod
• Reagents used:
– N/10 HCl
– Distilled water
• Procedure:
– Take N/10 HCl into the graduated Hb tube upto the 2 gm mark.
– Add 20cumm (0.02 ml) of blood using Hb pipette.
– The contents are mixed and allowed to stand for 10 minutes.
– The solution is then diluted using distilled water, stirred using
stirrer and compared with the glass standard.
• Advantages of this method:
a. Cheap and can be used for hemoglobin estimation where
automated hematology analyzer is not available.
b. Can be used for bedside Hb estimation
• Disadvantages of this method:
a. 95% colour of acid hematin is formed in 10 mins.
b. Carboxyhemoglobin, methemoglobin, and sulfhemoglobin are
not converted to acid hematin.
c. Acid hematin solution is not stable and color formation is slow
d. Source of light will influence the visual comparison of colors.
e. Color of brown glass standard fades with time.
f. Individual variation in matching of color is seen.
 Cyanmethemoglobin method
• Spectrophotometric method
• Internationally recommended method
• Principle: Blood is mixed in a solution
containing potassium cyanide and potassium
ferricyanide (Drabkin’s solution).
The potassium ferricyanide converts Hb to
methemoglobin which is converted to
cyanmethemoglobin by potassium cyanide.
The absorbance of the solution is then
measured in a spectrophotometer at a
 wavelength of 540nm.

• Advantages:
➢ The colour is stable so the readings need not be taken immediately.
➢ Almost all forms of Hb (except sulfhemoglobin) are converted to
cyanmethemoglobin.
➢ The method is accurately standardizable.
➢ Interperson variation not seen
➢ Standard solution of cyanmethemoglobin is very stable.
➢ Drabkin’s solution can be preserved for months.
 Thoma Red & White Blood Cell Diluting
Pipettes
They are diluting pipettes found in the Haemocytometer apparatus
They are used to dilute the blood to facilitate cell counting
 RBC And WBC Pipettes:Uses

WBC Pipette
The WBC
RBC Pipette
Pipette
• To
determin
How to differentiate?
- .

- -

- -

- -
Improved Neubauer’s chamber

It is a counting
chamber device
originally designed
and usually used for
counting blood cells
Improved Neubauer’s chamber
• It is a thick glass slide,
the centre of which has a
double ruling area
separated by troughs
 Why improved ?
Neubauer improved
• In the old chamber, the gap between the triple
lines was very wide
• Hence the rectangular space between them
looked similar to the squares in which cells
were counted
• This often resulted in erroneous counting
• These faults were corrected in the new
chamber, hence it is called improved
 Automated cell counters
• They are computerised,
highly specialised and
automated machines that
count the number of white
and red blood cells in a blood
cells
• They allow multiple tests in a
short period of time,
eliminate errors of human
nature, and allow for
counting of certain
parameters not possible to
calculate manually
 Principles of Automated cell counters
• Impedance (Coulters) principle: cell counting and sizing is
based on detection and measurement of changes in electrical
impedance produced by a particle as it passes through a small
aperture
• Optical Method: As each blood cell of a diluted blood specimen
passes through a sensing zone, it scatters a beam of laser light
focused on it, which is detected as an electrical impulse
• Flow cytometry: A beam of light is directed on a
hydrodynamically focused stream of fluid. Each suspended
particle scatters the light, and fluorescent chemicals attached to
it are excited into emitting light
• Selective lysis of red cells and counting of white cells
• Special stains
 Wintrobe and
Westergren Tubes
 Wintrobe and Westergren Tubes
• They are narrow glass tubes used for the measurement of
erythrocyte sedimentation rate (ESR)
• ESR is the the rate at which red blood cells in anticoagulant
whole blood descend in a standardized tube
• Stages of ESR
➢ Stage of rouleaux formation: 10 mins
➢ Stage of sedimentation: 40 mins
➢ Stage of packing: 10 mins
• Hence, measurement of ESR is done at end of the first hour
Wintrobe tube Westergren tube
• The tube is closed at lower end,
- . . .
• The tube is open at both ends
- .

open at upper
-

-
• It is 300 mm long, calibrated upto
• It is 110 mm long, calibrated upto 200 mm
100 mm • Bore diameter is 2.5 mm
• Bore diameter 3 mm • Anticoagulant used is sodium
- -

• Anticoagulant used is ammonium citrate (1:4 ratio)

-

potassium oxalate (3:2 ratio by • It can hold more volume of blood

weight) -
• It can hold less volume of blood
 ↑ 0 -9

⑭ 0-28
F

Wintrobe tube Westergren tube

• Uses:
Can be used to measure both ESR and
PCV
• Normal ESR Values:
Male: 0-9 mm at end of first hour
Female: 0-20 mm at end of first hour
• Normal PCV Values:
Male: 40-54%
Female: 36-47%
• Uses:
Can only be used to measure ESR (NOT
PCV)
• Normal ESR Values:
Male: 0-5 mm at end of first hour
Female: 0-7 mm at end of first hour
 Wintrobe tube Westergren tube
• Advantages • Advantages
➢ It requires less amount of blood ➢ It is a more sensitive and accurate
➢ It can also be used to measure PCV method
➢ Blood sedimentation results in the ➢ It provides better and more accurate
formation of 3 layers (each with dilution or blood
additional use)
1. Plasma: deep yellow colour indicates
jaundice
2. Buffy coat layer: may be used to
prepare smears is cases of low blood
cell count
3. Packed RBCs: used to measure PCV

Wintrobe tube Westergren tube

• Disadvantages • Disadvantages
➢ It is a less sensitive method ➢ Requires more amount of blood
➢ It cannot be used to measure PCV
 Other Methods
• Modified Westergren Method: It uses EDTA as the
anticoagulant over citrate
➢ It does not cause significant dilution of blood, avoiding the
errors
• MicroESR Method: It involves the use of microhematocrit
capillary tube to measure the distance that erythrocytes have
fallen after one hour in a vertical column of anticoagulant blood
under gravity
➢ It requires a small amount of blood (0.6 ml). It is preferable in
neonates
• Zeta Sedimentation Rate: It is similar to ESR; a capillary tube is

Pasteur Pipette

• A Pasteur pipette , also known as an

eye dropper, or dropper, is a device
used to transfer small quantities of
liquids.
• It has a narrow tip at one end and a
bulb at the other end
• It is mainly used to transfer blood
into the Wintrobe tube
 Bone Marrow Aspiration Needle/ Salah’s
Needle
• Parts of needle:

1. Trocar

2. Cannula
3. Adjustable side guard: It helps in
adjusting the depth of penetration
of the needle.
 Sites for BM aspiration
1. Iliac crest, ASIS, PSIS: Most common site for BM aspiration, but not
suitable in obese patients
2. Tibial tuberosity and iliac crest: in children
3. Sternum: Between 2nd and 3rd rib (manubrium can be used but contains
fat)
 Indications
• Absolute indications: • Relative indications:
1. Aplastic anemia 1. Refractory Anemia
2. Megaloblastic anemia 2. Thrombocytopenic Purpura
3. Acute leukemia 3. Infections: kala azar, malaria,
4. Myelofibrosis TB, histoplasmosis
5. Myelosclerosis 4. Secondary tumors
6. Multiple myeloma 5. Storage diseases: Gaucher's
disease, Nieman-Pick’s
disease
 Contraindications
• Hemophilia
 Dry Tap
• Dry Tap:
Failed aspiration or when marrow aspiration yields only blood or no
bone marrow particles at all.
• Causes of dry tap:
1. Faulty Technique -
2. Myelofibrosis -
3. Infiltration of tumor -
4. Severe hyperplasia (AML and Megaloblastic anemia)
5. Hypoplasia or aplasia of the marrow
Other Needles: Jamshidi’s needle/ Islam needle for BM trephine biopsy

BM Biopsy vs BM Aspiration:
Better BM architectural pattern is visualized in BM biopsy.
Cell morphology is better observed in BM aspiration.

Types of BM Biopsy:
Aspiration biopsy (complications: pain, local hematoma, fat embolism,
infection)
Percutaneous needle biopsy
Open surgical biopsy.
 Collection and Staining
• Collection:
Not more than 0.25 ml (to minimize dilution) periodic
said
Schiff
• Staining:
1. Romanowsky: Giemsa
2. Histochemical: peroxidase, PAS
3. Perl’s stain for iron stores
 What to look for in the smears?
1. Cellularity
2. Myeloid:erythroid ratio
3. Nature of erythropoiesis (normocytic/microcytic/megaloblastic)
4. Changes in megakaryocytes
5. Siderotic granules, iron stores in anemia
6. Abnormal cells: Gaucher’s cells, myeloma cells, malignant cells.
7. Parasites:- leishmania, histoplasma
 LIVER BIOPSY NEEDLE
(VIM SILVERMANN’S NEEDLE)
Bifid needle:
The shape is bifid as it helps for easy
removal of tissue specimen.
 Indications
• To know cause of: Hepatomegaly, Jaundice, Ascites, GIT Bleeding
• Differentiate between surgical and medical jaundice when LFT is inconclusive.
• Diagnosis of neoplasm
• Staging of Hodgkin's Lymphoma
• Systemic diseases: sarcoidosis, amyloidosis, TB
• Evaluating effectiveness of treatment
 Contraindications
• Bleeding disorder
• Suspected Hemangioma
• Hydatid Cyst
• Subphrenic Abscess
• Right sided empyema
• Severe anemia
 • Precautions:
1. Platelet count > 80,000/cmm
2. Prothrombin activity must be above 40% of normal

• Disadvantages:
3. False negative in 5-10 % (due to inadequate sample)
4. Bile peritonitis in patients with bile duct obstruction
5. Cannot differentiate between extra and intra hepatic cholestasis

• Other needles:
6. Menghini
7. Iverson Roholm needles
 LUMBAR PUNCTURE NEEDLE
• Collection:
1. Between L3 and L4
 Uses/Indications
• Diagnostic: Meningitis, Subarachnoid hemorrhage, Encephalitis, CNS Syphilis
• Therapeutic: Administer antimicrobial and anticancer drugs, relieve increased
ICT
• Introduce Anesthetics and Radiopaque Dyes
• Removal of exudates/ blood from Subarachnoid Space
 Contradictions
1. Idiopathic Increased ICP (may cause uncal herniation)
2. Bleeding disorder
3. Skin infection at puncture site
4. Vertebral deformities (scoliosis, kyphosis)
Urinometer
1. Used for measuring specific gravity of urine.
2. Specific gravity is directly proportional to concentration of solutes and
inversely proportional to volume of urine.
3. Normal range: 1.003 – 1.030
4. Temperature correction: Urinometer is calibrated at 15 Degree Celsius *for
every 3 degree Celsius rise/fall in temperature correction of 0.001 is to be
done
 • Increased in: DM, Nephritic Syndrome, Fever and dehydration
• Decreased in: Diabetes insipidus, chronic renal failure
• Isosthenuria:
1. Fixed specific gravity (1.010)
2. Seen in chronic renal disease- due to inability of concentration or
dilution of urine in CKD, i.e. Urine specific gravity = plasma specific
gravity.
The Process of Tissue Processing
Fixation

Dehydration

Clearing

Embedding

Sectioning

Staining
 FIXATION
• A process by which the constituents of cells and tissues are fixed in a
chemical or physical state, so that they can withstand the subsequent
treatment with different reagents with minimum loss , distortion or
decomposition.
• Principle-to denature or precipitate proteins to form a meshwork to hold
cell constituents.
• Types of fixation- Heat fixation, perfusion, immersion
Tissue processor
 Duration of fixation and size of tissue
• The fixative volume should be atleast 10 times the
volume of tissue specimen for optimal and rapid
fixation.
• For most fixatives the time of fixation is approximately equal to
the square of the distance which the fixative must penetrate.
• The gross specimens should not rest at the bottom of a
container of fixative and should be separated by soaked
paper or cloth.
• Unfixed gross specimens that are to be cut and stored in
fixative prior to processing should not be thicker than
 0.5cm.

FIXATIVES
Commonly used fixatives include-
1. 10% neutral formalin-which contains 40%formaldehyde (10ml) and distilled
water (90 ml)
2. Zenker's fluid
3. Bouin's fluid
4. Alcohol
5. Ether
6. Glutaraldehyde
 10% neutral formalin
• It is the most common fixative used
• Constituents-
a) Tap water=900ml
b) Formalin=100ml
c) sodium phosphate,monobasic, monohydrate=4g
d) Sodium phosphate,dibasic, anhydrous=6.5g
• pH should be 7.2 to 7.4
 10% neutral formalin

Advantages Disadvantages
Cheap, easily available and relatively Slow fixative
stable

Penetrates tissue reasonably Fumes might irritate

Does not cause excessive hardening Causes formation of pigments (Dark

brown pigments are formed in tissues
containing blood)

Frozen sections can be easily prepared

Staining for fat can be easily carried out
 Zenker’s fluid
• Potassium Dichromate and Mercuric Chloride
• Stains the tissue yellow
Advantages Disadvantages
Recommended for striated muscle Over hardening of tissue
No formation of pigmentation as seen in Sometimes excess pigment can be
formalin when used for specimens deposited.
containing blood; for example, spleen
infarcts.
Improves staining of connective tissue To remove excess mercury pigment:
Lugol’s iodine is used
To remove excess dichromate: washed
under running water
 Bouin’s fluid
• Constituents-Picric acid , formalin, acetic acid
• Preferred for testicular biopsy and smaller biopsies like that if GIT
• Used for demonstration of glycogen in tissues

Advantages Disadvantages
Penetrates rapidly, preserves glycogen Causes overhardening of tissue
and provides excellent staining
Explosive when dry so stored under H2O
 Other fixatives

• Alcohol-
• It is a powerful dehydrating agent.
• It precipitates glycogen
• It is the fixative of choice for demonstration of glycogen in tissues in cases of
liver biopsy in glycogen storage disease.
• Ether-
• used in cytological examination.
 Glutaraldehyde
• Glutaraldehyde causes extensive cross linking which results in
better preservation of ultra structure.
• It is used majorly for electron microscopy.Other fixatives used
for electron microscopy include-Carson modified Millonig
Formalin, Osmium tetroxide, aldehyde,etc.
• Disadvantage-it negatively affects the immunohistochemical
methods.
TISSUE CASSETTE
• Material used is either metal or
plastic

Uses:
1. For carrying the tissue
section after it has been fixed
Excess of fixative can be
filtered out through cassette
2. It is perforated to allow free
flow of chemicals
TISSUE TEK
• Material used is plastic
• It consists of 2 parts…
• A stainless steel base mold in
which tissue block is
embedded
• A plastic mold, placed on top
and filled with wax.
• Used for preparing paraffin
block with tissue.
L MOULD

• Leuckhart’s mould
• Material used is brass
• This material prevents
metal expansion
• Used for preparing
paraffin block with tissue
i.e. embedding of block
• Size – 10mm x 5mm x
5mm
PARAFFIN BLOCK HOLDER

• Also called as chuck

• Holds the paraffin block
(prepares using L mould)
• Attached to the microtome
for section cutting
TISSUE PARAFFIN BLOCK

Uses

• To enable tissue section

cutting with a microtome
• After microtome, section
thickness is 5 to 7
micrometers
GLASS SLIDE
WITH TISSUE
SECTION
GLASS SLIDE
A microscope slide is a thin flat
piece of glass, typically 75 by
26 mm (3 by 1 inches) and about
1 mm thick, used to hold objects
for examination under
a microscope. Typically the
object is mounted (secured) on
the slide, and then both are
mounted together on the
microscope for viewing
COVERSLIP
A thin glass plate used to
cover samples mounted on a
microscope slide.
 Frozen Section/Cryosection
• The Frozen Section/Cryosection
procedure is a pathological laboratory
procedure used to perform rapid
microscopic analysis of a specimen
• It is used most often in oncological
surgeries
• The key instrument for Cryosection is
the cryostat, which is essentially a
microtome inside a freezer
 Procedure of Cryosection
• The histology slide is cut at 5-10 micrometers
• The specimen is placed on a metal tissue disc which is then
secured in a chuck and frozen rapidly to about -20 to -30 deg
C
• The specimen is embedded in a medium called OCT
(containing ethylene glycol and polyvinyl alcohol
• At this temperature, most tissues become rock hard (all
tissues may have unique processing temperatures)
• Subsequently it is cut frozen with the microtome, and the
section is picked on a glass slide and stained
 Uses
• The principal use of a frozen section procedure is the examination
of tissue while surgery is taking place. It is a simple method for real
time margin control of a surgical specimen. It helps the surgeon
decide whether tumor resection is feasible

• Cryosection is also used to detect the presence of substances lost in

the traditional histology technique eg. Lipids, glycogen, etc
 Disadvantages

• Tissue morphology may be distorted

• Cellular details are not well seen
• Staining is not very good
• Some special stains cannot be performed
 CYTOLOGY
The study of the structure and function of cells

Applications:
1. Diagnosis and management of cancer
2. Intraoperative cytopathological consultation
3. Diagnosis of non-neoplastic/inflammatory conditions
4. Diagnosis of specific infections
5. Cytogenetics
6. Assessment of hormonal status in women
 Branches of cytology
Exfoliative cytology

1. Gynaecological smears - papanicolaou smear

2. Respiratory cytology
3. Urinary cytology
4. Body fluid cytology
5. Gastrointestinal tract samples

Aspiration cytology
6. FNAC
 FNAC
Fine needle aspiration cytology is a diagnostic procedure in
which a thin hollow needle is inserted into the mass for
sampling of cells which after being stained are examined
under the microscope.

Uses:
• For diagnosis , in masses and lesions
• To determine the benign or malignant nature of known
tumors
• To assess the treatment prognosis
 Methods of Obtaining Samples
1. Brushings
2. Punctures
a) Paracentesis
b) Thoracocentesis
3. Scrapings
4. Swabs
5. Washings Brushing Thoracocentesis

Methods of Smear Preparation

6. Streaking
7. Spreading
8. Pull apart
9. Touch Impression

Nasopharyngeal swab Bronchial Washing

 Staining Methods in Cytology

1. Papanicolaou Stain

2. May-Grunwald-Giemsea Stain

3. Nuclear Staining:- Hematoxilin Pap smear used for screening of

Cervical cancer and to evaluate the
hormone status of a woman.
Interpretation:-
4. Cytoplasmic Counterstaining:- Orange-G, Eosin Superficial SC indicate Estrogen
Azure levels (Cytoplasm stained red)
Intermediate SC indicate
Progesterone levels (Cytoplasm
stained blue-green)
Parebasal Cells indicate lack of E
and P Stimulation
Thank
You