BSc Laboratory Medicine Guide

BSc in Laboratory Medicine Part-II -1-
Edited by-
Monir Ahmed
B.Sc in Health Technology (Laboratory Medicine)
1st Batch
Institute of Health Technology
Mohakhali, Dhaka.
Monir Ahmed. BSc (Lab medicine), 1st batch, IHT, Dhaka. Mobile-01918-713432
email: monirahmed.bd@gmail.com web: http://a0720120505112739-8475723.WebStarts.com
Recommended for further reading-
1. Practical Pathology and Microbiology

- Prof. Kazi Khaleque, Prof. Kazi Mamun
2. A Short Text Book of Pathology
- Prof. Dr. Tahminur Rahman, Prof. Dr. Hosne Ara Tahmina
3. Review of Pathology and Genetics
- Gabind Rai Garg, Sparsh Gupta
4. Robins Pathologic Basis of Diseases.
5. A Text Book of Pathology
-N C Dey, T K Dey
6. Pathology Practical Book
-Harsh Mohan
7. de Gruchy’s Clinical Haematolgy in Medical Practice
- Frank Firkin, Colin Chesterman, David Penington, Bryan Rush
8. A Manual of Laboratory and Diagnostic Tests
- Frances Fischbach, Marshall B. Dunning III
9. Henry’s Clinical Diagnosis and Management by Laboratory Methods
- Richard A. McPherson, Matthew R. Pincus
10. Practical Pathology
- P. Chakaraborty, Gargi Chakraborty
11. Practical Pathology
-N C Dey, T K Dey, M Dey, D. Sinha
12. Essentials of Blood Banking
- SR Mehdi
13. Text Book of Transfusion Medicine
- Dr. Rana K Sherwani, Dr. Kafil Akhter
14. The Text Book of Blood Bank and Transfusion Medicine
- Satish Gupte
15. Hand book on Clinical Use of Blood- WHO, Geneva, Switzerland.
16. Medical Laboratory Technology (Vol-I,II,III)
- Kanai L. Mukherjee
17. Medical Laboratory Technology
- Ramnik Sood
18. A Concise Note on Medical Laboratory Technology
- C R Maiti
19. A Hand Book of Medical Laboratory Technology
- V.H. Talib
20. District Laboratory Practice in Tropical Countries (Part-1&2)
- Monica Cheesbrough
21. Blood Banking and Transfusion Medicine, Basic Principle and Practice
- Hillyer Silberstein, Anderson Roback
22. Differential Diagnosis in Laboratory Medicine
- Vincent Marks, Thomas Cantor, Dusan Mesko, Rudolf Pullman, Gabrieta N.
23. Medical Laboratory Technology
- Monir Ahmed
Science is organized knowledge. Wisdom is organized life. - Immanuel Kant
To my parents
Preface to the 2nd Edition
B.Sc in Health Technology (Laboratory Medicine) is a new diverse

and challenging subject. Yet the text and other study materials are not
available to all the students to have a strong grip on the subject. My small
effort may help the students to have a quick glance to the vast subjects such
as General pathology, Clinical pathology, Haematology, Blood Banking,
Histopathology and Cytopathology. I welcome any suggestions and
constructive criticisms from all the students and Medical Technologists alike to
enrich the guide in future.
I thank the Almighty Allah for the publication of this 2nd Edition
overcoming all the odds and hope all the students of BSc in Laboratory
Medicine may find this concise handbook helpful.
Last but not the least I must acknowledge my humble gratitude to my
following colleagues for their support and encouragement-
Sayed Mahmudul Hasan, MT (Lab), Shahid Suhrawardy
Medical College Hospital, Dhaka and Student of BSc in Lab
medicine, IMT, Dhaka.
MD. Shahidul Islam, MT (Lab), Mymensingh Medical College
and student of BSc in Lab medicine, 3rd batch, IHT, Dhaka.
Md. Rafiqul Islam, MT (Lab), IEDCR, DGHS, Mohakhali,
Dhaka and student of BSc in Lab medicine, Bangladesh Institute
of Child Health, Sher-e-Bangla Nagar, Dhaka.
All the student of Lab Medicine, 1st Batch, IHT, Dhaka.
The Editor
Contents
General Pathology
***Q- 1. Define cell injury. What are the physical agents causes cell injury? Write the pathogenesis
of ischaemic cell injury.
**Q-2. Define and classify inflammation. Give the difference between acute and chronic
inflammation.
*Q-3. Define & classify hypertrophy and hyperplasia. Write the difference between hypertrophy
and hyperplasia.
*Q- 4. Define & classify cell injury and adaptation. Describe the cellular adaptation.
Q- 5. Define & classify wound and wound healing? What are the factors influencing wound
healing? Give the complication of wound healing.
***Q-6. Define and classify tumour (neoplasm). Give the difference between benign and malignant
tumour.
**Q-7. Define and classify shock. Give the pathogenesis/mechanism of hypovolaemic and septic
shock.
***Q- 8. Define & classify infarction and oedema. Give the difference between thrombus and
embolus.
Q- 9. Define & classify thrombus and embolus? Write down the complication of thrombus and
embolus.
Q- 10. Define & classify Gangrene? Give the laboratory diagnosis of a tumour (or Cancer).
*Q-11. Define & classify necrosis. Write down the difference between necrosis & apoptosis.
Q- 12. What is ischaemia and what are its causes? Name the pathogenesis of thrombosis.
Q- 13. What are the causes of cell injury? State the cellular and tissue responses to different types
of cell injury.
Q- 14. Define exudate & transudate. Write the difference between exudate and transudate.
Q- 15. Write short notes on- *** i. Tumour markers.
ii. Apoptosis.
*** iii. Repair.
iv. Paraneoplastic syndrome.
v. Ulcer
Clinical Pathology
***Q- 1. Write down the routine examination of urine. Enumerate the clinical importance of urine
examination (routine examination of urine).
**Q- 2. What are the liver function tests? Give the indication of liver function tests.
***Q-3. Name the common ova found in stool with figure. Write down the indication of stool
culture.
**Q-4. Write down the indication, contraindication and complication of lumber puncture.
***Q-5. What are the indications of semen analysis? Write normal findings of semen analysis (or,
Write routine examination of semen with normal findings).
***Q-6. Define proteinuria and write down their causes. How can you estimate/detect protein in
urine?
*Q-7. How do you collect urine for laboratory tests? Name the reducing substance present in urine.
**Q-8. Name the common cysts & trophozoites found in stool with figures. Write the indication of
routine examination of stool. Write down the procedure of collection of stool for examination in the
laboratory?
***Q-9. Define glycosuria and write down their causes. How can you detect glucose in urine?
*Q-10. Mention the difference between bacterial, viral and tubercular meningitis.
Q- 11. Write the routine examination of CSF with the normal findings.
Q- 12. What is ketonuria and what are the causes? How can you detect ketone bodies in urine?
Q- 13. How bile salts, bile pigments and urobilinogen can be detected in urine? What is their
clinical significance?
Q- 14. Write down the principle and procedure of Gram’s stain and Ziehl- Neelsen’s stain.
Q- 15. Write short notes on: *** i. OBT.
ii. OGTT.
*** iii. Bence Jones protein.
iv. CPK
v. Body fluids
vi. Haematuria and Haemoglobinuria.
Information is not knowledge. It's not that I'm so smart, it's just that I
stay with problems longer.- Albert Einstein
Haematology
***Q- 1. Define erythropoiesis. What are the sites and stages of erythropoiesis? Name the factors
affecting erythropoiesis.
**Q- 2. Define and classify anticoagulants with examples. What are the anticoagulants commonly
used in the laboratory? Why EDTA is best?
*Q-3. What are the criteria of a good film? Write down the steps of preparation of a blood film.
***Q-4. Define MCH, MCV and MCHC with their normal values.
**Q-5. What are the causes of iron deficiency anaemia? Give the laboratory diagnosis of iron
deficiency anaemia.
***Q-6. Define and classify leukaemia. Give the laboratory diagnosis of acute myeloid leukaemia
(AML) and chronic myeloid leukaemia (CML) or, Give the bone marrow findings of AML and
CML.
**Q-7. Give the uses of Improve Neubauer Counting Chamber, Wintrobe tube, RBC & WBC
pipette.
***Q-8. Give the total count, differential count and absolute count of WBC. Draw and label WBCs.
Q- 9. Define PCV. Mention the causes of high and low PCV.
Q- 10. Name the methods of Haemoglobin estimation. Give the advantages and disadvantages of
Acid Hematin Method.
Q- 11. What is leukaemoid reaction? Give the differences between leukaemia and leukaemoid
reaction.
**Q-12. Define and classify anaemia. What are the causes of anaemia?
*Q-13. What is ESR? Give the normal values of ESR. Mention the causes of high and low ESR.
Describe the procedure of estimation of ESR by Westergren method.
**Q- 14. What is thrombocytopenia? What are the causes of thrombocytopenia?
Q- 15. Short notes on: i. APTT.
*ii. Polycythaemia.
*iii. Pancytopenia.
*** iv. MCV.
v. Myeloblast and lymphoblast
vi. Bone marrow aspiration
vii. Laboratory diagnosis of ITP
Blood Transfusion
***Q-1. Define blood group. Name the important blood groups. Why ABO blood group is called
classical blood group?
***Q-2. What are the methods of blood grouping? Describe the slide method of blood grouping.
**Q-3. Give the indication and contraindication of blood transfusion. What do you mean by Safe
Blood Transfusion?
Q- 4. Give the pathogenesis, treatment and prevention of Erthroblastosis foetalis.
***Q-5. Give the hazards/complications of blood transfusion.
***Q-6. Name the different plasma products. Give the indication, contraindication and risk of fresh
frozen plasma.
***Q-7. What are the types of platelet concentrate? Give the indication and contraindication of
platelet transfusion.
Q- 8. What is blood bank? What are the importance of a blood bank?
*Q-9. Name the blood constituents available for clinical use. Give the immune complication of
blood transfusion.
**Q-10. Give the indication and contraindication of human albumin.
*Q-11. Give the indication and contraindication of hydroxyethyl starch.
Q- 12. Name the methods of detecting antibody in the laboratory. Describe anyone method.
Q- 13. Give the composition, indication and contraindication of SAG mannitol blood.
Q- 14. How do you screen a blood donor? What are the tests done for screening of blood for
transfusion?
Q- 15. Write short notes on: ***i. Auto-transfusion.
***ii. HLA system.
***iii. Cross matching
*iv. Cryoprecipitate.
v. Natural and immune antibodies.
Short saying often contains much wisdom. - Sophocles
Histopathology and Cytopathology

*Q- 1. Define pneumonia. Give the classification and organisms of pneumonia. Give the stages of
lobar pneumonia/ Give the morphology of various stages of lobar pneumonia.
***Q-2. Define peptic ulcer (disease). Give the site and complication of peptic ulcer (disease).
***Q-3. How do you diagnose tuberculosis in the laboratory? (Or, Give the laboratory diagnosis of
Tuberculosis).
***Q-4. Define IHD. What are the risk factor and complication of MI?
***Q-5. Describe the aetiology and microscopic features of acute appendicitis.
***Q-6. Define nephrotic syndrome. Give the difference between AGN & nephrotic syndrome.
***Q-7. Name the intestinal ulcer. Give the difference between typhoid & tubercular ulcer.
**Q-8. What is cirrhosis of liver? Classify cirrhosis of liver. Give the pathogenesis and effects of
cirrhosis of liver.
**Q- 9. Define jaundice. Give the laboratory diagnosis of different types of jaundice (haemolytic &
obstructive jaundice).
*Q-10. Write down the risk factors, sites and classification of carcinoma of breast.
*Q-11. Give the aetiology, stages & pathogenesis of carcinoma of cervix.
***Q-12. Give the advantages, disadvantages and indication of FNAC.
***Q-13. How will you prepare a slide for histological examination?
Q- 14. Define AGN. Write down the pathogenesis of AGN.
*Q- 15. What are the sites of tuberculosis? Give the difference between primary & secondary
tuberculosis/Give the difference between primary & post primary tuberculosis.
Q- 16. Give the laboratory diagnosis of carcinoma of cervix and breast.
Q- 17. Enumerate the causes of rheumatoid arthritis. State the pathology of rheumatoid arthritis.
Q- 18. Define atherosclerosis. Write down the risk factors and complications of atherosclerosis.
*Q- 19. Give the pathogenesis & pathology of infective hepatitis with laboratory diagnosis.
Q-20. Briefly describe the Haemotoxylin and Eosin stain. Shortly describe the progressive and
regressive stain.
Q-21. Name five cytopathological specimens that are commonly received in the laboratorirs. How
will you process pleural fluid sample for cytopathological examination.
Q-22. Define and classify fixative. Which one is ideal fixative for biopsy specimen and why? What
are the equipments required for gross examination of a large soft tissue tumour.
Q-23. Name the malignant tumours of uterine cervix. Which test is done for carcinoma cervix.
Q-24. Define microtomy. What are the types of microtome? How will you care it?
Q-25. Mention the principle of tissue processing. What are the clearing agents? What are the
criteria for choosing an ideal clearing agent?
Q-26. Give the laboratory investigation of nephritic syndrome. Give the steps of PAP stain.
Q- 27. Write short notes on: i. Fibroadenoma of breast
**ii. Goiter
iii. Primary complex
iv. Biopsy
v. BCG vaccine
vi. Lymphoma
vii. Bronchogenic carcinoma
* viii. Gall stone
ix. Angina pectoris
x. Pleural effusion
xi. Diabetic ketoacidosis
xii. Oedema
xiii. Malignant cell
xiv. Shock
xv. Hyperplasia
xvi. Granuloma
xvii. Frozen section
xviii. Immunohistochemistry
xix. Decalcification
xx. Special stain
xxi. Quality control in histopathology laboratory
xxii. BEP
General Pathology
***Q- 1. Define cell injury. What are the physical agents causes cell injury? Write the
pathogenesis of ischaemic cell injury.
Answer. Definition of Cell injury: It may be defined as, “any adverse influence which
derange the cell’s ability to maintain a steady normal or adaptive homeostasis.” So, cell
injury results when the cell is exposed to an injurious agent or stress. It can also be defined
as, “the sequence of events that follows if the limits of adaptive response to a stimulus are
exceeded or if the cells are exposed to injurious agents or stress, deprived of essential
nutrients, or become compromised by mutations that affect the essential cellular
constituents.”
Physical agents that causes cell injury:
a. Mechanical trauma- May cause a subtle but significant dislocation or intracellular
organization of organelles or may destroy the cell by completely disrupting it.
b. Low temperature-
i. Induces vasoconstriction and impairs blood supply to cells →Hypoxia → cell
injury.
ii. If hypothermia persists for prolonged period → injury to vasomotor control with
marked vasodilatation, stagnation of blood flow and intravascular coagulation and when
temperature become significantly low, intercellular water crystallizes.
c. High temperature- causes hypermetabolism that results in:
i. exceeding the capacity of available blood supply
ii. accumulation of acid metabolites → ↓PH to critical level.
d. Sudden changes in atmospheric pressure- Deep sea divers or tunnel diggers. When
working under ↑ atmospheric pressure have higher level of atmospheric gases
dissolved in their blood. If such individual return to normal pressure too quickly,
the dissolved gases come out of solution rapidly and form air bubbles within the
circulation. O2 is easily re-dissolved but N2 is less soluble and may persist as small
bubbles that become trapped in the microcirculation, blocking the blood flow and
ultimately causing injury to cells. This condition is called caisson disease.
e. Electric shock- i. Produce heat → burn
ii. It may interfere in neural conduction pathways and often causes
death from cardiac arrhythmia.
f. Radiation- It causes cell injury by:
i. Direct ionization of chemical compounds contained within the cell.
ii. Ionization of cellular water producing free hot radicals that secondarily interact
with intercellular constituents.
iii. Induces varying mutations that may injure and even kill cells.
Pathogenesis/Mechanism of ischaemic or hypoxic cell injury:
Hypoxia
Reversible cell injury Irreversible cell injury (Cell death)
Membrane injury → i. Loss of phospholipids
Ischaemia ii. Reactive oxygen species (free radical)
iii. Cytoskeletal abnormality
Mitochondria iv. Lipid breakdown products
↓
↓ Oxidative phosphorylation Ca2+ influx → Ca 2+ in mitochondria
Cell swelling Leakage of enzymes (CK, LHD)
↓ATP ↓ Na Influx of Ca2+ Loss of microvilli
pump ↑Na+, H2O ER swelling Lysosomal enzyme ↓ Basophilia ( ↓ RNP)
Efflux of K+ Myelin figures release and activation Nuclear changes
Blebs within the cell Protein digestion
↑Glycolysis ↓ Glycogen
↓ PH
Clumping of
nuclear chromatin
ER= Endoplasmic reticulum
Detachment of RNP= Ribonucleoprotein
ribosomes ↓Protein synthesis
↓
Lipid deposition
**Q-2. Define and classify inflammation. Give the difference between acute and chronic
inflammation.
Answer. Definition of inflammation:
a. Inflammation is the reaction of tissues to all forms of injury.
b. Reaction of vascularized tissue to injury is called inflammation.
c. Inflammation is a complex reaction in living vascularized tissue to injurious agents
leading to the exudation and accumulation of protein rich fluid and leukocytes in
extravascular tissue, provided the injury does not destroy the tissue.
Classification of inflammation: According to duration, inflammations are of 2

types-
a. Acute inflammation: It is an immediate and early response to an injurious
agent and lasts for minutes to a few days. It is an exudative lesion.
Morphologically, acute inflammations are of following types-
i. Serous: e.g. Pleural effusion
ii. Fibrous: e.g. Pericarditis
iii. Purulent: e.g. Abscess, boil.
iv. Ulcer
b. Chronic inflammation: It is an inflammation that persists for weeks to

months. It is a proliferative lesion. It is again subdivided into following
categories-
i. Chronic non-specific inflammation, e.g. Chronic osteomyelitis
ii. Chronic specific inflammation, e.g. Tuberculosis.
Difference between acute and chronic inflammation:

Trait Acute inflammation Chronic inflammation
1. Common causes Pyogenic bacteria Tubercle bacilli, Lepra bacilli
2. Duration (usual) Minutes to days Weeks to months
3. Character Exudative Proliferative, Granuloma may form
4. Vascular phenomenon Prominent Not prominent
5. Fluid exudate Marked Usually scanty
6. Cellular exudate Early neutrophils then Mononuclear cells in most cases
macrophages in most cases
7. Repair process Follows Starts concurrently
8. Type Stereotypic Non-stereotypic
9. Example Acute appendicitis Tuberculosis, Leprosy, Syphilis
*Q-3. Define & classify hypertrophy and hyperplasia. Write the difference between
hypertrophy and hyperplasia.
Answer. Definition of hypertrophy:
a. Increase in the size of the cells leading to increase in the size of the organ is called
hypertrophy.
b. Hypertrophy is the increase in the size of the cells resulting in an increase in the
size of the organ.
c. Hypertrophy is the increase in the size of an organ or tissue due to increase in the
size of its constituent specialized cells.
Causes of hypertrophy: a. Increased functional demand.

b. Specific hormonal stimulation
Types of hypertrophy: A. Physiological, e.g. Muscles in athlete, Uterus in

pregnancy, breast during lactation due to prolactin and oestrogen.
B. Pathological, e.g. Left ventricular hypertrophy in
hypertension, aortic stenosis due to increased workload.
Definition of hyperplasia:
a. Increase in the number of cells in an organ or tissue with increase in volume is
called hyperplasia.
b. Hyperplasia is an increase in the number of cells in an organ or tissue, which then
may increase in volume.
BSc in Laboratory Medicine Part-II - 10 -
c. Hyperplasia is an increase in the size of an organ or tissue due to an increase in

number of its specialized constituent cells.
Causes of hyperplasia:
a. Induction of different genes encoding GF (growth factor), GFR (Growth
factor receptor).
b. Increased local production of GF, GFR.
c. Results in cellular proliferation. Hormone may act as growth factor.
Types of Hyperplasia: A. Physiological-

a. Hormonal hyperplasia, e.g. Development of female breast during
puberty or pregnancy
b. Compensatory hyperplasia, e.g. hyperplasia of liver after partial
hepatectomy.
B. Pathological-
a. Prostate- benign hyperplasia in old age
b. Male breast- gynaecomastia.
c. Endocrine glands- parathyroid hyperplasia in chronic renal failure.
Difference between hypertrophy and hyperplasia:

Traits Hypertrophy Hyperplasia
1. Definition Increase in the size of the cells Increase in the number of cells
leading to increase in the size in an organ or tissue with
of the organ is called increase in volume is called
hypertrophy. hyperplasia.
2. Source of stimuli Mechanical / Hormonal Chemical or Hormonal.
3. Types of cells involved Permanent cells Labile or stable cells.
4. Growth cycle Occurs after G2 phase. No Occurs in G2 phase. Mitosis
mitosis. occurs.
5. Malignant transformation Does not occur Pathological hyperplasia is a
fertile soil for malignant
transformation.
6. Example Hypertrophy of skeletal Benign hyperplasia of prostate.
muscle. Hyperplasia of endometrium.
Hypertrophy of cardiac muscle.
*Q- 4. Define & classify cell injury and adaptation. Describe the cellular adaptation.
Answer. Definition of Cell injury: It may be defined as, “any adverse influence which
derange the cell’s ability to maintain a steady normal or adaptive homeostasis.” So, cell
injury results when the cell is exposed to an injurious agent or stress. It can also be defined
as, “ the sequence of events that follows if the limits of adaptive response to a stimulus are
exceeded, or if the cells are exposed to injurious agents or stress, deprived of essential
nutrients, or become compromised by mutations that affect the essential cellular
constituents.”
Classification of cell injury: A. According to severity-

1. Reversible / mild cell injury: Morphological changes
resulting from non-lethal injury to cells. Cells become
normal when injurious stimuli are withdrawn.
2. Irreversible/ severe cell injury: Morphological changes that
follow cell death in a living tissue or organ. The cells can not
come to its original form after removal of injurious agent.
B. Morphological types-
1. Reversible cell injury: a. Cellular swelling
b. Fatty changes
2. Irreversible cell injury: a. Necrosis
b. Apoptosis
Definition of Adaptation /cellular adaptation: It may be defined as, “the ability of

the cells to adapt and achieve an altered but steady state, preserving the health of the cell
despite continued stress.” It can also be defined as, “the reversible functional and structural
responses to more severe physiologic stresses and some pathologic stimuli, during which
new but altered states are achieved, allowing the cell to survive and continue to function.”
Types of cellular adaptation:

I. Atrophy- Atrophy is the decrease in cell size.
II. Hypertrophy- Hypertrophy is the increase in cell size.
III. Hyperplasia- Hyperplasia is the increase in cell number.
IV. Metaplasia- Metaplasia is the change in the type of the cell.
Description of different types of cellular adaptation:
I. Atrophy- Shrinkage in the size of the cell by loss of cellular substance is known as
atrophy.
Types of Atrophy:
1. Physiological, e.g. on old age brain and sexual organ and in puberty-
lymphoid tissue & thymus.
2. Pathological,
a. Localized atrophy-
i. Ischaemic atrophy, e.g. cerebral atheroma
ii. Pressure atrophy, e.g. capsule of benign tumour.
iii. Disuse atrophy, e.g. muscles, ligaments, bone atrophy due to
immobilization of the joints.
iv. Neuropathic atrophy, e.g. wasting of paralyzed limbs in motor
(lower) neuron disease, poliomyelitis etc.
v. Idiopathic atrophy, e.g. myopathies, motor neuron disease etc.
b. Generalized atrophy-
i. Starvation atrophy
ii. Senile atrophy
iii. Endocrine atrophy (hypopituitarism)
iv. Osteoporosis (bone atrophy)
II. Hypertrophy- It refers to an increase in the size of the cells, resulting in an increase in
the size of the organ.
Types of hypertrophy:
1. Physiological, e.g. Uterus in pregnancy
2. Pathological, e.g. aortic stenosis due to increased workload.
III. Hyperplasia- It is an increase in the number cells in an organ or tissue, usually

resulting in increase mass of organ or tissue.
Types of Hyperplasia:
1. Physiological, e.g. Glandular tissue in female breast.
2. Pathological, e.g. Skin warts due to papilloma virus.
IV. Metaplasia- It is a reversible change in which one adult cell type (epithelial or
mesenchymal) is replaced by another adult cell type. It can also be defined as, “an
adaptive transformation of one adult cell type by another cell type of similar tissue.
Types of Metaplasia:
1. Epithelial metaplasia-
⇒ Squamous metaplasia, e.g. benign enlargement of prostate.
⇒ Columnar metaplasia. e.g. intestinal variant of gastric
carcinoma.
2. Connective tissue metaplasia, e.g. myositis ossificans.
3. Tumour metaplasia, e.g. carcinoma of the gall bladder.
Q- 5. Define & classify wound and wound healing? What are the factors influencing
wound healing? Give the complication of wound healing.
Answer. Wound: A wound is a break in the continuity of skin of the body. It can also be
defined as, “a breach in the superficial tissue continuum”.
Types of wound-
a. Incised wounds: These are caused by sharp instruments like knife, razor etc.
The blood vessels are clear cut and so these wounds bleed very much.
b. Contused wound: These are caused by blows by blunt instruments or by
crushing. The tissues are bruised.
c. Lacerated wounds: These are caused by machinery, falls on rough surfaces

places of shells, claw of animals etc. These wounds have torn and irregular
edges and they bleed less.
d. Punctured wounds: These are caused by stabs by any shape instruments like
knife or dagger or needle. They have small opening but may be very deep.
Wound healing: a. Healing or wound healing is the replacement of damaged,

degenerated or lost tissue or cell by viable tissue or cell of similar or dissimilar character.
b. It is the replacement of destroyed cells or tissues by the
proliferation of viable cells either by similar cells (regeneration) or by fibroproliferative
response with scar formation (repair) or by fibrosis.
Types of wound healing:
1. Primary healing/ Healing by first intention (primary union)
2. Secondary healing/ Healing by second intention (secondary union)
Difference between Primary and Secondary Union:
Primary Union Secondary Union
1. Clean incised (surgical wound) 1. Lacerated wound.
2. Wound edges are closed to each other, 2. Edges are wide apart, wide no gap.
no gap.
3. Minimum tissue damage. 3. Maximum tissue damage.
Primary Union Secondary Union
4. Less necrotic cells. 4. More necrotic cells.
5. Little exudate. 5. More exudate.
6. Infection usually absent. 6. Infection usually present.
7. Little bleeding. 7. More bleeding.
8. Less granulation. 8. More granulation tissue formation.
9. Rapid healing. 9. Delayed healing.
10. Minimum or less tissue scar. 10. Maximum scar tissue.
11. Less complication. 11. More complication.
12. Wound contraction absent. 12. Wound contraction present.
13. No loss of specialization function. 13. Some loss of specialization function.
Factors influencing wound healing:

A. Local factors-
1. Blood supply – poor ↓, rich ↑
2. Adhesion to bony surface ↓
3. Tissue tension ↓
4. Local infection ↓
5. Foreign body/ soft tissue interposition ↓
6. Local movement ↓
7. Ionizing radiation ↓
8. Ultraviolet light ↓
B. General factors-
1. Age- old age ↓, early age ↑
2. Nutrition-
a. Malnutrition ↓
b. Vitamin C deficiency ↓
c. Protein deficiency ↓
d. Vitamin D- necessary to repair of fracture.
3. Systemic diseases
a. Diabetes
b. Malignant disease Delay wound healing.
c. Uraemia
d. Severe anaemia
e. Jaundice
Complication of wound healing:
1. Infection- delay wound healing
2. Wound bursting or rupture.
3. Implantation cyst- epithelial cells which flow into the healing wound may sometime
persists and proliferate to form cyst.
4. Keloid formation- it is a raised area of scar tissue due to excessive formation of

collagenous tissue.
5. Painful scar- if neuroma is included in scar tissue.
6. Weak scar- incisional scar.
7. Pigmentary changes- due to deposition of excess haemosiderin.
8. Cicatrisation- excessive reduction in the size of the scar.
9. Metaplasia- cartilage and bone formation may occur.
10. Neoplasm- squamous cell carcinoma may develop.
11. Hyperplasia.
***Q-6. Define and classify tumour (neoplasm). Give the difference between benign and
malignant tumour.
Answer. Definition of neoplasm: Neoplasm means new growth. Tumour is equated with
neoplasm. Willis definition of neoplasm states that, “a neoplasm (tumour) is an abnormal
mass of tissue, the growth of which exceeds and is uncoordinated with that of normal
tissues and persists in the same excessive manner after cessation of the stimuli which
evoked the change.”
Classification of tumour: I. Classification on behaviour- Tumours are of
following two type: 1. Benign tumours- these are mostly harmless, e.g. lipoma, osteoma.
2. Malignant tumours- Malignant tumour may be of carcinoma
(tumour of epithelial tissues) or sarcoma (tumours of connective tissue/ mesenchymal
origin). e.g. Fibrosarcoma, liposarcoma, osteogenic sarcoma. Cancer is the term for all
malignant tumours.
II. Classification on tissue of origin-
1. Epithelial tumours: tumours of epithelial cells.
2. Mesenchymal tumours: tumours of mesenchymal cells.
3. Mixed tumours: more than one neoplastic cell type.
4. Tumours of totipotent cells: Teratomas.
Difference between benign and malignant tumours:

Characteristics Benign tumours Malignant tumours
1. Differentiation/anaplasia 1. Well differentiated. 1. Anaplastic, well to poorly
differentiated, or undifferentiated.
2. Pleomorphism (variation in 2. Absent 2. Often present.
size & shape) of neoplastic cells
3. Orientation of neoplastic cells 3. Not distributed. 3. Distributed.
4. Nucleus 4. Not hyperchromatic, not 4. Hyperchromatic, pleomorphic, nuclei
pleomorphic, nucleus is little are usually large, mitotic figures are
altered; mitotic figures are few usually numerous and abnormal.
and normal.
5. Nucleus/ cytoplasm ratio 5. Normal. 5. Increased.
6. Rate of growth 6. Slow, progressive 6. Rapid, expansile.
7. Size 7. Usually small. 7. Usually large.
8. Stroma 8. Well formed. 8. Usually poorly formed.
9. Haemorrhage and necrosis 9. Little tendency. 9. Common.
10. Capsule 10. Usually encapsulated. 10. Not-capsulated.
11. Local invasion 11. Expansive but not invasive 11. Invasive.
12. Clinical effects & fatality 12. Usually not fatal. 12. Almost invariably fatal.
13. Removal 13. May be easily removed. 13. Complete removal often impossible.
14. Recurrence 14. Little tendency to recur. 14. Recurrence is common.
**Q-7. Define and classify shock. Give the pathogenesis/mechanism of hypovolaemic and
septic shock.
Answer. Shock:
a. Shock may be defined as, “widespread hypoperfusion of tissue due to
reduction in the blood volume or cardiac output or redistribution of blood
resulting in an inadequate effective circulatory volume.”
b. Shock is a disorder that results from systemic hypoperfusion due to
reduction either in cardiac output or in the effective circulatory blood
volume.
Classification of shock:
a. Cardiogenic shock, e.g. Myocardial infarction, Ventricular rupture, Arrhythmia,
Cardiac temponade, Pulmonary embolism.
b. Hypovolaemic shock, e.g. Haemorrhage, fluid loss (vomiting, diarrhoea, burns,

trauma).
c. Septic shock, e.g. overwhelming microbial infection, endotoxic shock, Gram
positive septicaemia, Fungal species, Super antigen.
d. Neurogenic shock, e.g. Anaesthetic accident, spinal injury.
e. Anaphylactic shock, e.g. Generalized IgE mediated hypersensitivity reaction
(Type-I hypersensitivity).
Aetiology of septic shock/ endotoxic shock:
1. Endotoxin producing Gram- negative bacteria (Esch. coli, Proteus, Pseudomonas).
2. Some Gram-positive bacteria (Streptococci, Pneumococci).
3. Some fungi.
Pathogenesis of septic shock:

1. Endotoxin and exotoxin are produced after infection by organisms producing them.
↓
2. They form a complex with lipopolysaccharide (LPS) binding protein in the serum.
↓
3. The complex binds to CD14, receptors on leukocytes (especially monocytes &
macrophages).
↓
4. Initiation of synthesis, release or activation of cascade of mediators derived form
plasma cells or cells (monocytes, macrophages, endothelial cells etc).
↓
5. Endogenous mediator-
a. Cytokines, e.g. tumour necrosis factor (TNF), IL- 1,2,6,8, nitric oxide,
platelet activating factor, endorphins.
b. Arahidonic acid metabolites (prostaglandins & leukotrienes), complement
C5a, kinin, coagulation factor, myocardial depressor substance.
↓
6. Action of mediators on different organs of the body-
a. Heart: Diminished myocardial contractility.
b. Blood vessels: Systemic vasodilatation (hypotension), thrombosis, DIC.
c. Lungs: Acute respiratory distress syndrome (ARDS).
d. Generalized organ dysfunction.
↓
Septic shock
Pathogenesis of haemorrhagic/ hypovolaemic shock:
A. Reversible shock-
Haemorrhage, severe burn, severe vomiting & diarrhoea.
↓
Acute hypovolaemic shock
↓
Decreased central venous pressure.
↓
Decreased cardiac filling (end diastolic volume)
↓
Decreased cardiac output
↓
Decreased arterial pressure
↓
Reversible shock
↓
Compensatory autoregulation
↓
Increased arterial pressure.
B. Irreversible shock-
Haemorrhage, severe burn, severe vomiting & diarrhoea.
↓
Severe hypovolaemia
↓ If not corrected
Compensatory mechanism failure
↓
Hypoperfusion of tissues
↓
Acute hypoxia
↓
i. Injury to endothelium, that leads to activation of coagulation cascade and ultimately
DIC (Disseminated intravascular coagulation)
ii. Anaerobic glycolysis, leads to decreased PH which leads to vasodilatation and
ultimately fall in blood pressure.
↓
Severe hypoxia
↓
Damage to cells and tissues
↓
Acute renal tubular necrosis
↓
Complete renal shutdown
↓
Death
***Q- 8. Define & classify infarction and oedema. Give the difference between thrombus
and embolus.
Answer. Infarction: Infract is an area of ischaemic necrosis caused by occlusion of either
the arterial supply or the venous drainage in a particular tissue. The process of formation of
an infract is known as infraction.
Classification of infraction:
A. On the basis of colour of infract-
1. Red/ Haemorrhagic infract.
2. White/pale/ anaemic infract.
B. On the basis of bacterial contamination-
1. Bland infract: bacterial infection absent.
2. Septic infract: bacterial infection present.
C. On the basis of duration-
1. Recent infraction
2. Old infraction
Oedema: 1. Oedema may be defined as, “an excessive extravascular accumulation of

fluid.”
2. Accumulation of excess fluid in the interstitial tissue space or body
cavities.
Classification of oedema: I. On extent of involvement-
1. Localized oedema, e.g. pulmonary oedema, ascities,
lymphoedema, inflammatory oedema.
2. Generalized oedema, e.g. cardiac oedema, renal oedema, hepatic
oedema, nutritional oedema.
II. On Pathophysiology-
1. Inflammatory oedema
2. Non-inflammatory oedema.
III. On the nature of fluid-
1. Exudate
2. Transudate
IV. On clinical basis-
1. Pitting oedema
2. Non-pitting oedema
Difference between thrombus and embolus:

Traits Thrombus/ Thrombi Embolus/Emboli
1. Definition 1. It is a solid or semi-solid mass
1. An embolus is a detached
formed within the cardiovascular intravascular solid, liquid or gaseous
system from the constituents of the
mass that is carried by the blood to a
streaming blood. site distant from its point of origin.
2. Composition 2. Constituents of streaming blood.
2. Constituents of streaming blood, or
other substances, e.g. air, fat etc.
3. Site 3. Remains at the site of origin. 3. Travels away from the site of
origin.
4. Attachment 4. Attached to the vessel wall. 4. Free floating.
Traits Thrombus/ Thrombi Embolus/Emboli
5. Consistency 5. Solid or semi-solid. 5. Solid, liquid or gaseous.
6. Clinical 6. Less dangerous in comparison 6. More dangerous.
importance to embolus.
Q- 9. Define & classify thrombus and embolus? Write down the complication of thrombus
and embolus.
Answer. Definition of thrombus: 1. It is a solid or semi-solid mass formed within from the
cardiovascular system from the constituents of streaming blood.
2. Thrombus is a solid mass formed within noninterrupted
cardiovascular system from the constituents of the blood particularly platelets during life.
Thrombosis is the formation of solid mass in the circulation from the constituents of
the streaming blood. The mass itself is called a thrombus and consists of aggregated
platelets and fibrin in which red cells and white cells are trapped.
Classification of thrombus:
A. According to site-
1. Arterial thrombosis
2. Venous thrombosis:
a. Phlebothrombosis.
b. Thrombophebitis
c. Other types
3. Thrombosis in the heart:
a. Vegetations
b. Mural thrombosis
c. Laminated thrombus
d. Ball thrombus
B. Based on infection-
1. Bland/ aseptic thrombus, e.g. coronary thrombi
2. Septic/ infected thrombus, e.g. vegetation of infective
endocarditis.
C. Based on composition & colour-
1. Pale thrombus: platelets only (white)
2. Red thrombus: composed mainly by blood clots e.g. occluded
thrombus.
3. Mixed thrombus: composed of platelets, fibrin and clot, e.g.
coralline thrombus.
Definition of Embolus: 1. Embolus is a detached intravascular solid, liquid or

gaseous mass that is carried by the blood to a site distant form its point of origin and the
process of embolus formation is called embolism.
2. Embolus is an intravascular solid, liquid or gaseous mass that
is carried to a site distant from its point of origin.
Embolism is the travel of embolus from its site of origin and resulting in impaction
with partial or complete occlusion of a vessel too small to permit its further passage.
Embolism may be defined as, “occlusion of some part of the cardiovascular system by
impaction of a foreign mass or embolus transported to the site through the blood stream.”
Classification of embolus:
A. According to physical state-
1. Solid: a. Thrombo-embolic (99%)
b. Bone marrow embolism
c. Tumour cell embolism
d. Foreign body embolism, e.g. bullets, silk, talc.

e. Parasites, e.g. Schistosomes.
f. Red cell aggregation in infection & trauma.
g. Micro-emboli.
2. Liquid: a. Fat embolism

b. Amniotic fluid embolism.
3. Gaseous: a. Air embolism
B. According to location-
1. Arterial
2. Venous
3. Lymphatic
C. According to presence or absence of infection-

1. Bland or aseptic
2. Septic or infected
D. Miscellaneous-
5. Paradoxical: Embolus that arises in the venous circulation but
enters the arterial site or vice versa.
6. Retrograde embolus: that travels against the flow of blood.
Complication of thrombosis/thrombi: The thrombi are of clinical importance for

two reasons- 1. They cause obstruction of arteries and veins.
2. They provide possible source of emboli. The effects of thrombosis
depends upon: a. Size and shape of the thrombus
b. Degree of vascular occlusion
c. Kind of vessel invaded.
d. Degree of collateral circulation
e. Organ involved.
The possible effects/ complications are-
1. No effect if thrombus is small or lysed.
2. Effects at the site of thrombus:
a. Cord like thickening
b. Redness
c. Pain
d. Tenderness
e. Infection
3. Effects distal to the site:
a. Vein- i. Dilated and tortuous.
ii. Oedema.
b. Artery- i. Gangrene (necrosis of tissues with superadded
putrefaction).
ii. Ischaemia and infarction
4. Proximal to the site: Embolism
5. Infection
6. Post thrombotic ulceration
7. Death (Massive pulmonary embolism)
Complications/Main effects of emboli (or, embolism) are:

1. Infection
2. Gangrene- by bland emboli
3. Abscess- by septic emboli
4. No effect if the emboli is small
5. Secondary metastases by tumour emboli
6. Sudden death- when large vessels are involved or emboli large in
size.
7. Functional disturbance.
Q- 10. Define & classify Gangrene? Give the laboratory diagnosis of a tumour (or
Cancer).
Answer. Gangrene: It can be defined as, “necrosis of tissue with superadded putrefaction”.
It can also be define as, “macroscopic death of tissue followed by desiccation or
putrefaction.”
Classification of Gangrene: Gangrene are divided into-
1. Dry gangrene: It occurs due to lack of arterial blood supply.
2. Moist gangrene: It develops due to obstruction of both arterial and venous
drainage particularly in moist area.
3. Gas gangrene (Clostridial myonecrosis): It is caused by anaerobic Clostridium
perfringens (Cl. welchii) in most cases, and rarely by Cl. novyi and Cl.
septicum.
Laboratory Diagnosis of a tumour (or, Cancer):

1. Clinical history
2. Past history- Family history, Medical history
3. Biochemical tests- Tumour associated marker or enzymes.
4. FNAC- any palpable mass either external or internal (CT/USG guided)
5. Cytology- PAP’s smear, any body fluid, any washing or brushing, lavage, any
discharge (from nipple, sinus, pus etc).
6. Histopathology- Biopsy/resection of tumour, Lumpectomy, Frozen section.
7. Special stain- like pus for glycogen producing tumours, Mucin for mucin
producing tumours, Sudan black blue or Oil red O for lipid, Reticulin for reticulin
fibers, Masson’s Trichrome for fibroblast and collagen, T & B cell markers.
8. IHC- detection of specific antigen by specific antibody.
9. Immunofluorescence (IF) study- specially to see antigen- antibody deposit in
skin lesions, kidney disease.
10. Genetic studies- Chromosome analysis, karyotyping, HLA and tissue typing,
amniotic fluid analysis.
11. Molecular diagnosis- PCR, FISH technique.
12. Electron microscopy- specially to see organelles changes, sub-epithelial, sub-
endothelial and mesangial deposits in kidney diseases, BM thickening or
abnormality.
13. Tissue and cell culture
14. Stem cell culture.
*Q-11. Define & classify necrosis. Write down the difference between necrosis &
apoptosis.
Answer: Definition of Necrosis: 1. Necrosis is the sum of the morphological changes that
follow cell death in a living tissue or organ.
2. It can be defined as, “sum of morphological changes in a
dying cell in living tissue due to degradative action of lysosomal enzymes.”
Classification of necrosis: A. Basic type/Main type-
1. Coagulative necrosis (found in Heart, kidney, liver etc).
2. Liquefactive necrosis (found in Central Nervous System).
B. Special types-
a. Caseous necrosis (e.g. T.B)
b. Fat necrosis: i. Enzymatic- e.g. in Pancreas
ii. Traumatic- e.g. in Breast
c. Gangrenous necrosis
d. Fibrinoid necrosis, e.g. certain collagen disease, Rheumatic fever, SLE,
Polyarteritis nodosa.
e. Gummatous necrosis, e.g. Tertiary syphilis.
f. Necrosis of muscle, e.g. Zenker’s degeneration
g. Osteonecrosis
Difference between necrosis and apoptosis:

Condition Necrosis Apoptosis/Necrobiosis
1. Cause 1. Pathologic invariably 1. Physiologic and Pathologic
2. Pathogenesis 2. ATP depletion, membrane 2. Endonuclease, Gene
injury, Free radical damage. activation.
3. RNA break down 3. Random, diffuse. 3. Internucleosomal.
4. Cell size 4. Enlarged 4. Reduced.
5. Nucleus 5. 5. Fragmented. Pyknosis.
Pyknosis→Karyorrhexis→
Karyolysis.
6. Plasma membrane 6. Disrupted. 6. Intact.
7. Cellular content 7. Enzymatic digestion 7. Intact. Apoptotic bodies-
phagocytosis.
8. Adjacent inflammation 8. Frequent. 8. No inflammation.
9. Cell involved 9. Large group of cells 9. Single cell or small group of
cells.
10. Genetic predisposition 10. Absent. 10. Present.
11. Enzyme involved 11. Lysosomal enzyme. 11. Endonuclease and Caspases.
Q- 12. What is ischaemia and what are its causes? Name the pathogenesis of thrombosis.
Answer. Ischaemia: Ischaemia may be defined as, “inadequate supply of blood to an area
of tissue.”
Ischaemia produces harmful effects in 3 ways- i. Hypoxia
ii. Malnutrition
iii. Failure to remove waste products.
Causes of ischaemia:
A. General causes-
1. Inadequate cardiac output.
2. Complete heart block
3. Ventricular arrest or fibrillation
B. Local causes-
1. Arterial obstruction by thrombosis, embolism, spasm, atheroma etc.
2. Venous obstruction by venous thrombosis, strangulated hernia, volvulus etc.
3. Obstruction of small vessels by vasculitis, arthus reaction, severe cold (spasm)
etc.
Effects of ischaemia: 1. No effect if adequate collateral circulation exists.

2. Functional disturbances – Angina pectoris
3. Degeneration- Necrosis, Fibrosis.
The pathogenesis of thrombosis: Virchow's triad describes the pathogenesis of

thrombus formation which includes the following three predisposing factors-
a. Endothelial injury (injury to the endothelial cells that line enclosed spaces of the
body, such as the inside of blood vessels) e.g. trauma, atheroma.
b. Abnormal blood flow (loss of laminar flow resulting from stasis in veins or
turbulence in arteries) (e.g. valvulitis, aneurysm).
c. Hypercoagulability e.g. leukaemia, Factor V mutation (Leiden).
Q- 13. What are the causes of cell injury? State the cellular and tissue responses to different
types of cell injury.
Answer. Causes of cell injury:
1. Oxygen-
a. Hypoxia: A cause of hypoxia is inadequate oxygenation of blood in cardiac
failure and respiratory failure.
b. Ischaemia, i.e. inadequate blood supply to an area due to diminished arterial flow
or reduced venous drainage, e.g. in atherosclerosis, thrombosis, embolism. Ischaemia is the
most common cause of hypoxia.
c. Inadequate oxygen-carrying capacity of blood, e.g. anaemia, carbon monoxide
poisoning.
2. Infectious agents: Bacteria, Viruses, Fungi, Protozoa and Helminthes.
3. Immunological reactions: Hypersensitivity reaction causes cell injury.
4. Physical agents: Mechanical trauma, burn, ionizing radiation, cold.
5. Drug and chemical agents: Alcohol, narcotics, cytotoxic drugs, poisons.

6. Nutritional imbalances: e.g. Protein energy malnutrition, excess of lipids,
excess of food leading to obesity.
7. Genetic defects, e.g. inborn errors of metabolism.
Cellular and tissue responses to different types of injury: Cellular responses to
injury varies in its effects on cell structure and function according to the type of cell
involved and the nature and severity of the agent responsible. They include-
1. Cellular adaptation: a. Atrophy

b. Hypertrophy
c. Hyperplasia
d. Metaplasia
2. Cell injury: a. Acute reversible cell injury
b. Irreversible injury/Death-
i. Necrosis
ii. Apoptosis
c. Subcellular alterations in various organelles.
3. Intercellular accumulations
4. Pathological calcification
5. Cellular aging.
Q- 14. Define exudate & transudate. Write the difference between exudate and transudate.
Answer. Exudate: It is an inflammatory extravascular fluid that has a high protein
concentration (>3gm/dl), containing cellular debris and has a high specific gravity (above
1.020).
Transudate: It is the fluid formed in non-inflammatory oedema with a low protein
content (mostly albumin) and with a little or no cellular material resulting in low specific
gravity.
Exudation: The escape of fluid, protein and blood cells from the vascular system
into the interstitial tissue or body cavities is known as exudation.
Difference between exudate and transudate:
Features Exudate Transudate
1. Cause 1. Increased vascular permeability. 1. Increased hydrostatic
pressure.
2. Gross appearance 2. High coloured and turbid. 2. Clear and transparent, may
be yellow.
3. Nature of fluid 3. Inflammatory 3. Non-inflammatory.
4. Types of protein 4. As in plasma 4. Mostly albumin.
5. Total protein 5. High, more than 3 gm/dl. 5. Low, less than 1gm/dl.
6. Fibrinogen 6. Present, so it clots spontaneously. 6. Not present, no spontaneous
coagulation.
7. Seromucin 7. Present. 7. Absent.
8. Specific gravity 8. High, over 1020. 8. Low, below 1006 to 1012.
9. Cells 9. Plenty polymorphs or 9. A few cells may be present.
lymphocytes.
10. Bacteria 10. Usually present. 10. Absent.
Q- 15. Write short notes on- *** i. Tumour markers.
ii. Apoptosis.
*** iii. Repair.
iv. Paraneoplastic syndrome.
v. Ulcer
Answer.
*** i. Tumour markers: Tumour markers are substances, usually proteins that are
produced by the body in response to cancer growth or by the cancer tissue itself. Some
tumour markers are specific for one type of cancer, while others are seen in several cancer
types. Many of well known markers are seen in benign non-cancerous conditions as well as
in cancer.
So, tumour markers are biochemical indicators of the presence of a tumour. They
are detected in- plasma, stool, body fluids, urine and tissues.
Tumour markers may be-

a. Cell surface antigens
b. Cytoplasmic proteins, e.g. immunoglobulins, PSA, and PSMA
c. Enzymes, e.g. PAP, NSE.
d. Hormones, e.g. HCG, Calcitonin, Catecholamines, ectopic hormones.
Some common selected tumour markers and associated conditions-
Markers Associated cancers

1. Hormones
◊ Human chorionic gonadotrophin Trophoblastic tumours, nonseminomatous
(HCG) testicular tumours.
◊ Calcitonin Medullary carcinoma of thyroid
◊ Catecholamine and metabolites Pheochromocytoma and related tumours.
◊ Ectopic hormones Paraneoplastic syndromes.
2. Oncofetal antigens
◊ α- fetoprotein Live cell cancer, nonseminomatous germ
cell tumour of testis.
◊ Carcinoembryonic antigen Carcinoma of colon, pancreas, lungs,
stomach and heart.
3. Isoenzymes
◊ Prostatic acid phosphatase Prostate cancer
(PAP)
◊ Neuron specific enolase Small cell cancer of lungs, neuroblastoma.
4. Specific proteins
◊ Immunoglobulins Multiple myeloma and gammopathies.
◊ Prostate specific antigen (PSA) Prostate cancer
5. Mucins and other
glycoproteins
◊ CA -125 Ovarian cancer
◊ CA- 19-9 Colon cancer, pancreatic cancer.
◊ CA- 15-3 Breast cancer
6. New molecular markers
◊ p53, APC, RAS mutations in Colon cancer
stool and serum
◊ p53 and RAS mutations in stool Pancreatic cancer
and serum
◊ p53 and RAS mutations in Lung cancer
sputum and serum
◊ p53 mutations in urine Bladder cancer.
ii. Apoptosis (or, necrobiosis): Apoptosis is a distinctive morphologic pattern of cell

death. It is also known as programmed cell death where cell is destined to die due to
genetically determined programmed death. A single cell or a small group of cells is
destroyed. Apoptosis can be activated by death triggering signals like a lack of growth
factor or hormone, specific injurious agent, positive ligand- gland interaction. Apoptosis is
important in the regulation of density of normal cell population. Suppression of cell death
by apoptosis is a determinant of the growth of cancer. Apoptosis mean “falling off.”
Causes of apoptosis- Apoptosis occurs under physiological and pathological
situations:
A. Physiological situation, example:
1. During embryogenesis- by programmed destruction of cells, e.g. implantation,
developmetal involution.
2. Cell deletion during proliferation, e.g. intestinal crypt epithelia.
3. Hormone dependent involution in adult, e.g. regression of lactating breast after
weaning, prostatic atrophy after castration.
B. Pathological cause, example:
1. Death of neutrophils in acute inflammatory responses.
2. Death of immune cells, e.g. death of B and T lymphocyte at the end of immune
response.
3. Cytotoxic T cell induced cell death.
4. Viral diseases, e.g. Viral hepatitis, in which apoptotic cell fragments in the liver
are termed Councilman bodies.
5. Injurious agents, which can cause necrosis, produce apoptosis in low doses, e.g.
cytotoxic anticancer drugs, radiation, mild thermal injury and hypoxia.
6. Cell death in tumour.
7. Pathological atrophy after duct obstruction, e.g. Parathyroid gland, pancreas.
8. Pathological atrophy of hormone dependent tissues, e.g. Loss of lymphocytes in
the thymus after glucocorticoid administration.
Morphology/Morphological features of Apoptosis-

1. Cell shrinkage.
2. Chromatin condensation: It is the most characteristic feature of apoptosis.
3. Formation of cytoplasmic blebs and apoptotic bodies.
4. Phagocytosis of apoptotic cells or cell bodies by parenchymal cell or
macrophages.
Significance/ Importance of Apoptosis-
The process is important physiologically in balancing cell proliferation and
elimination. It is associated with: 1. Maintenance of organ size in adult.
2. Organ development and modeling in the embryo.
3. Physiological hypertrophy of involution.
*** iii. Repair: Repair can be defined as, “the process of replacement of dead or
damaged tissues by new healthy cells (parenchymal or stromal). Repair is the replacement
of injured or dead cells after injury like inflammation, wounds, surgical resection by
proliferation of viable cells. The goal of the repair process is to restore the tissue to its
original state. It signifies restitution of the part to a (more or less) complete replica or
template or image of its former structure.
Repair occurs by two distinct process- regeneration which restores normal tissues
and healing which leads to scar formation and fibrosis. Most commonly repair occurs by a
combination of these two processes.
Repair begins early in inflammation sometimes in 24 hours after injury.
iv. Paraneoplastic syndrome: A paraneoplastic syndrome is a disease or symptom

that is the consequence of the presence of cancer in the body, but is not due to the local
presence of cancer cells. These phenomena are mediated by humoral factors (by hormones
or cytokines) excreted by tumor cells or by an immune response against the tumor.
Paraneoplastic syndromes are typical among middle aged to older patients, and they most
commonly present with cancers of the lung, breast, ovaries or lymphatic system (a
lymphoma). Sometimes the symptoms of paraneoplastic syndromes show even before the
diagnosis of a malignancy.
This syndrome refers to a large group of complications and medical problems in

patients who suffer from cancer. These conditions are not due to the direct impact of the
tumors, yet rather due to the production of chemical substances from the cancer cells. The
chemical substances are produced and released in the blood stream and their effect may
present itself in various ways. Not all cancers cause Paraneoplastic Syndrome.
Paraneoplastic syndrome indicates occult neoplasm, in affected patient this may
produce significant clinical problem and may even be fatal, mimic metastasis and confound
treatment. Most of these are due to elaboration of ectopic hormones produced by these
malignancies. Commonly elaborated hormones are-
a. ACTH or ACTH like substances in small cell carcinoma of lung.
b. Production of PTH in SSC of lung causinh hyperparathyroidism.
c. Clubbing of fingers in lung cancer, the cause of which is unknown.
d. Venous thrombosis (known as Trousseau’s sign) found in pancreatic carcinoma,
lung carcinoma due to hypercoagulability.
e. Polycythaemia in renal cell carcinoma patients due to elaboration of
erythropoietin.
v. Ulcer: Ulcer is a local defect or excavation due to loss of continuity of

epithelial surface of an organ or tissue that is produced by the sloughing (shedding) of
necrotic tissue.
Types of Ulcer-
1. Inflammatory Ulcer (or, Benign ulcer):
a. Non-specific ulcer, e.g. ulcer due to pressure and injury
b. Specific ulcer, e.g. ulcer due to tuberculosis, syphilis.
2. Malignant ulcers: Ulcerative malignant tumours, e.g. basal cell
carcinoma, squamous cell carcinoma.
vi. Nomenclature of tumours:

Tissue of Origin Benign Malignant
A. Epithelial tumours Papilloma and Adenoma Carcoma
1. Stratified squamous Squamous cell papilloma Squamous cell carcinoma
epithelium
2. Basal cell of epidermis of - Basal cell carcinoma
skin or adnexa
3. Transitional epithelium of Transitional cell papilloma Transitional cell carcinoma
urinary tract
4. Glandular or ductal Adenoma, Cystadeonoma, Adenocarcinoma,
epithelium Paiplloma Cystadeonocarcinoma,
Papillary carcinoma
B. Mesenchymal tumours - Sarcoma
1. Fibroblast Fibroma Fibrosarcoma
2. Fat cell Lipoma Liposarcoma
3. Smooth muscle Leiomyoma Leiomyosarcoma
4. Blood vessels Haemangioma Angiosarcoma
5. Striated muscle Rhabdomyoma Rhabdomyosarcoma
6. Lymph vessels Lymphangioma Lymphangiosarcoma
7. Bone Osteoma, Osteosarcoma.
Giant cell tumour of bone.
8. Cartilage Chondroma Chondrosarcoma
9. Brain coverings Maningioma Malignant meningioma
10. Haemopoietic stem cell - Leukaemia
11. Lymphoid cell - Lymphocytic leukaemia and
lymphoma.
C. Mixed tumours - -
1. Breast Fibroadenoma -
2. Salivary gland Mixed tumours of salivary Malignant mixed tumour of
gland salivary gland
3. Kidney - Wilms’ tumour
D. Tumour of Totipotent Mature or benign teratoma Immature or malignant
cells teratoma
Our prime purpose in this life is to help others. And if you can't help them,
at least don't hurt them. - Tenzin Gyatso
Clinical Pathology
***Q- 1. Write down the routine examination of urine. Enumerate the clinical importance
of urine examination (routine examination of urine).
Answer: Routine examination of urine involves: A. Physical examination
B. Chemical examination
C. Microscopic examination.
A. Physical examination-
1. Quantity: For a normal adult the urine output is about 700-2500ml/day depending
largely on the fluid intake.
2. Colour: Normally light straw.
3. Appearance: Normally clear and transparent.
4. Sediment: Absent in normal urine.
5. Specific gravity: Normal urine has a specific gravity in between 1010 to 1020.
B. Chemical examination-
1. Reaction: Normal urine is usually acidic.
2. Protein: Normal urine may contain upto 150mg protein (mostly albumin) in 24
hours which does not give a positive boiling test (which require ≥ 500mg/ml).
3. Reducing substances (e.g. sugar): Normally absent.
4. Ketone bodies: Normally absent.
5. Bilirubin: Normal urine contains no bilirubin.
6. Urobilinogen: A small amount of urobilinogen is excreted in normal urine.
7. Bile salts and bile pigment: Normally absent.
8. Blood: Absent in normal urine.
9. Chyle: Normally it is not present in urine.
C. Microscopic examination-
1. In a centrifuge take a suitable amount of urine (5-10ml).
2. Centrifuge it at 1500-3000 rpm for 5-10 minutes.
3. Decant the supernatant urine from the centrifuge tube.
4. Re-suspend the sediment and place a drop of sediment on a clean glass slide.
5. Place a cover slip over it and examine it under microscope. First observe the
deposits under low power lens and then confirm it under high power lens.
6. The following things are usually observed under the microscope:
a. Cells- A few epithelial cells and a very few leukocytes (pus cells) are
found in normal urine. RBCs are not found in normal urine.
b. Cellular Casts (epithelial cast, RBC cast, WBC cast)- Normally absent.
c. Other casts (hyaline cast, granular cast, waxy cast)- Normally absent.
d. Bacteria and Parasites- Normally absent.
e. Spermatozoa and others- Normally absent.
Clinical importance of urine examination:

A. Clinical importance of routine examination of urine-
1. Renal and urinary tract diseases, e.g. urinary tract infection (UTI), nephrotic
syndrome, acute glomerulonephritis (AGN) etc.
2. Metabolic or systemic diseases, e.g. Diabetes mellitus, Diabetes insipidus,
Jaundice, Acidosis etc.
B. Clinical significance of some special examination of urine-
1. Cultural examination of urine for the diagnosis of enteric fever, infection of
kidney and urinary tract etc.
2. Some biochemical analysis of urine as a renal function test, e.g. urea clearance
test, creatinine clearance test etc.
3. Exfoliative cytology to detect neoplasm in urinary tract.
4. In male, lesions of prostate and seminal vesicle.
5. To detect calculi (stone) in urinary tract.
6. Detection of HCG in urine as a diagnosis of pregnancy.
7. To follow up the treatment with anti-diuretic drugs.
8. Some special narcotic drug tests, e.g. heroin, morphine.
9. Detection of tumour markers in urine, e.g. PSA (prostate specific antigen).
**Q- 2. What are the liver function tests? Give the indication of liver function tests.
Answer. Liver function tests (LFT): The tests which are used to assess the condition or
disease of liver.
A. Pigment metabolism-
1. Blood: a. Serum Bilirubin
b. Vanden Bergh’s reaction/ Vanden Bergh’s test.
c. Icteric index
2. Urine: a. Bile salts

b. Bile pigment
3. Stool: a. Stercobilinogen
b. Bilirubin
B. Enzymatic functions
1. SGPT (Serum glutamic pyruvic transaminase)/ALT (Alanine
aminotransferase)
2. SGOT (Serum glutamic oxaloacetic transaminase)/AST (Asparate
aminotransferase)
3. LDH (Lactic dehydrogenase)
4. Alkaline phosphatase
5. γ glutaryl transferase.
6. Serum isocitrate dehydrogenase.
C. Synthetic activity-
1. Serum albumin
2. Serum albumin globulin ratio
3. Prothrombin time
4. Clotting time and APTT (activated partial thromboplastin time)
D. Metabolic activities-
1. Carbohydrate metabolism: a. Glucose tolerance test
2. Protein metabolism: a. Serum total protein
3. Fat & lipid metabolism: a. Serum total cholesterol
b. Stool for faecal fat
c. Serum Triglycerides
d. Cholesterol ester
e. Abnormal lipoproteins
E. Detoxification activity-
1. Blood ammonia level
2. Hippuric acid test
F. Excretion of foreign substance-
1. Bromsulphthalein test/ Bromsulphalein (Sulfobromophthalein) clearance
test
2. Rose Bengal test
G. Serological tests-
1. Mitochondrial antibody
2. Antinuclear and smooth muscle antibody
3. Viral antigens and antibodies:
a. Hepatitis A virus
b. Hepatitis B virus
c. Hepatitis C virus
d. Hepatitis D virus/ Delta virus
e. Hepatitis E virus
H. Liver biopsy
I. Miscellaneous -
1. Serum α fetoprotein
2. Serum ferritin level.
3. Total iron binding capacity
4. Serum α1- antitrypsin
Indication of Liver function test:

1. Differential diagnosis of jaundice
2. To diagnose liver damage
3. To assess the extent of liver damage
4. To follow up the progress of liver damage

5. To detect any malignancy of liver.
***Q-3. Name the common ova found in stool with figure. Write down the indication of
stool culture.
Answer: The common ova found in the stool are as follows-
Ankylostoma duodenale AL (Fertilized ova) AL (unfertilized ova) Trichuris trichiura
Enterobious vermicularis Taenia spp. Hymenolepsis nana
Indication of stool culture: 1. Cholera and diarrhoeal diseases-

a. Cholera: caused by V. cholerae.
b. Diarrhoea: caused by Escherichia coli
c. Dysentery: caused by Shigella
2. Enteric fever-
a. Typhoid and paratyphoid fever: caused by Salmonella spp.
3. Food poisoning-
a. Non-toxic type: caused by Salmonella spp, Campylobacter
jejuni, Bacillus cereus.
b. Toxic type: caused by Staphylococcus aureus, Clostridium
botulinum, Clostridium perfringens, Escherichia coli.
**Q-4. Write down the indication, contraindication and complication of lumber puncture.
Answer. Indication of lumber puncture/ indication of CSF collection:
A. Diagnostic purposes-
a. Diagnosis of –
a. Meningitis
b. Meningoencephalitis
c. Encephalitis
d. Brain abscess
e. Subarachnoid haemorrhage
f. Leukaemia
g. Multiple myeloma
h. Gullain Barre Syndrome
i. Spinal cord tumours
j. Intracranial haemorrhage due to injury
k. Intracranial occupying lesions.
b. Differential diagnosis of Cerebral infract versus intracerebral haemorrhage
(80% CSF show Xanthomatosis in later cases).
B. Therapeutic purposes-
a. Relief from intracranial pressure
b. Removal of exudate or blood from sub-arachnoid space
c. Introduction of serum, e.g. anit-meningococcus, anti-tetanus.
d. Introduction of drugs, e.g. hypertonic solution of MgSO4, NaCl.
e. Spinal anaesthesia, e.g. Novocain, eucaine etc.
f. Introduction of contrast media or isotopes, e.g. myelography.
Contraindication of lumber puncture:

a. Brain tumour at cerebello-pontine angle or in region of 3rd ventricle
associated.
b. Spinal cord tumour.
c. A recent intracranial haemorrhage which may be reactivated by the
disturbance of the clot.
d. Brain abscess that may possibly spread.
e. Septicaemias
f. Puncture should never be done when patient is in convulsion.
g. Advance cardiovascular diseases including that of arteries.
h. Any feature of raised intracranial pressure, e.g. papilloedema.
i. Depressed level of consciousness.
j. Any bleeding disorders, e.g. thrombocytopenia.
Complication of lumber puncture:

1. Herniation of uncus through tentorium or cerebellar tonsils through
foramen magnum.
2. Paresis to paralysis due to Spinal Cord Trauma.
3. Extradural or Subdural haematoma with resultant paraplegia.
4. In presence of sepsis, perforation of the meninges.
5. Introduction of infection by passing the needle through superficial or
deep sepsis in the lumber region.
6. Post puncture headache.
7. In infants death due to asphyxia.
***Q-5. What are the indications of semen analysis? Write normal findings of semen
analysis (or, Write routine examination of semen with normal findings).
Answer. Indication of semen analysis:
a. To diagnose male sterility.
b. Medicolegal purposes, e.g. suspected rape.
c. To investigate the effectiveness of vasectomy and of recanalizatin operation.
Semen: It is a milky viscid liquid ejaculated by male containing the secretion of
testes as well as male accessory reproductive organs (e.g. prostate, urethra, epididymides
etc).
Composition of semen:
A. Secretion from testes- 05%
a. Spermatozoa
B. Secretion from seminal vesicles- 60%
a. Fructose (1.5-6.5mg/ml)
b. Phosphorycholine
c. Argothioneine
d. Ascorbic acid
e. Flavins
f. Prostaglandins
C. Secretion from prostate- 20%
a. Spermine
b. Citric acid
c. Cholesterol, phospholipids
d. Fibrinolysin, fibrinogenase
e. Zinc
f. Acid phosphatase
D. Others- 15%
a. Secretion from Epididymides
b. Secretion from Vasa deferentia
c. Secretion from Bulbourethral and Urethral glands
Normal findings of semen analysis (Routine examination of semen with normal

findings):
A. Physical examination/ Naked eye examination-
1. Volume: At least 0.5ml/ejaculation and on average normally 2 – 5 ml per
ejaculation.
2. Colour: Normal semen is white in colour.
3. Consistency: Normally turbid and viscid.

4. Odour: Fishy odour.
5. Specific gravity: Normally on an average 1.028.
6. Liquefaction: Normally occurs within 10-30 minutes.
1. PH: Normally 7.2-8.0 (slightly alkaline).
2. Fructose: Normally fructose content is 1.5-6.5 mg/ml.
3. Acid Phosphate: Normal semen has about 2500 KA units/ml of acid phosphate.
C. Microscopic examination-
1. Total spermatozoa: Normally 40-100 million/ml.
2. Motility:
a. Normally at the end of 1st hour more than 80% spermatozoae are motile.
b. Normally at the end of 2nd hour more than 60% spermatozoae are motile.
c. Normally at the end of 4th hour more than 40% spermatozoae are motile.
3. Morphology: Normally more than 80% spermatozoa should have normal
morphology, i.e. a head, a neck and a tail.
***Q-6. Define proteinuria and write down their causes. How can you estimate/detect
protein in urine?
Answer. Proteinuria: The presence of protein in urine which gives a positive boiling test or
stix test is called proteinuria.
Causes of proteinuria:
A. Physiological-
1. Newborn upto ten days
2. Prolonged standing (orthostatic)
3. Heavy Exercise
4. Pregnancy
B. Pathological-
a. Renal diseases (Heavy/massive proteinuria > 3gm/day):
1. Nephrotic syndrome
2. Nephritic syndrome
3. Acute glomerulonephritis
4. Infections, e.g. Pyelonephritis, tuberculosis.
5. Toxaemia of pregnancy
6. Haematuria due to calculi, tumours.
b. Post-renal diseases (Moderate proteinuria > 1gm/day):
1. Severe urinary tract infections, e.g. Prostitis, urethritis.
2. Calculi of ureters and urinary bladder
c. Systemic or Pre-renal diseases (Mild proteinuria ≤ 1gm/day):
1. Febrile illness
2. Congestive heart failure
3. Hypertension
4. Systemic disorders associated with haematuria.
Detection/estimation of protein/ albumin in urine:

A. Qualitative tests –
1. Heat and acetic acid test / Heat coagulation test
2. Sulphosalicylic acid test
3. Heller’s test
4. Reagent strip method
B. Quantitative tests-
1. Esbach’s albuminometer method
2. Turbidimetric method
3. Urinary autoanalyzer method
*Q-7. How do you collect urine for laboratory tests? Name the reducing substance present
in urine.
Answer. Collection of urine for laboratory tests:
A. Collection of urine for routine examination-
1. Urine should be collected in a clean, dry container with well fitted tips.
2. A clean catch, mid-stream urine void at any time of the day but a morning specimen
is preferable as because the components are present in a concentrated form.
3. Usually 10-20 ml urine is collect, but for complete examination 50-100ml
sample is required.
4. Urine should be examined within 30 minutes of collection, if immediate
examination is not possible preserve the specimen by refrigeration at 2°-4°C
(usually it can be preserved for 2-4 hours).
B. Collection of urine for culture-

a. For male:
1. Clean thoroughly the glans penis with profuse water and soap and then dry.
2. Collect about 10-20 ml of urine from the mid-stream in a sterile container.
3. Plug the mouth of the container and transport it to the laboratory.
b. For female:
1. Wash the ano-vaginal area thoroughly with soap and water and then dry.
2. Apply antiseptics over the vulva.
3. Separate the vulva with thumb and finger and then micturate.
4. Hold the sterile container with the other hand and collect about 10-20ml of
urine from mid-stream.
5. Plug the container and transport it to the laboratory.
C. 24 hours urine sample-
1. At a specific time usually 08.00 hours, instruct the patient to empty his or her
bladder. Discard this urine.
2. Give the patient a large (3 liter capacity) bottle or container containing a
suitable preservative or stabilizer.
3. Instruct the patient to collect into the container all the urine he or she has passed
in the next 24 hours upto and including the urine passed at 08.00 hours
following the next morning.
D. Post-prandial sample-
1. Specimen taken 2 hours after the meal.
E. Random sample-
1. Sample taken at any time of the day.
Reducing substances present in urine:

Non- reducing substances present
a. Glucose
in urine:
b. Lactose
a. Bile salt
c. Fructose Sugar
b. Bile pigment
d. Galactose
c. Protein
e. Pentose
d. Ketone bodies
f. Sucrose
e. Urobilinogen
g. Homogenestic acid Non- Sugar
h. Uric acid and creatinine
**Q-8. Name the common cysts & trophozoites found in stool with figures. Write the
indication of routine examination of stool. Write down the procedure of collection of stool
for examination in the laboratory?
Answer. The commonly found cysts and trophozoites are of:
1. Entamoeba histolytica
2. Giardia lamblia
Indication of stool examination: Examination of stool is indicated in following

conditions-
1. Parasitic infestation
2. Dysentery
3. Jaundice
4. Cystic disease of pancreas
5. Ulcerative colitis
6. Coeliac diseases
7. Indigestion and malabsorption
8. Gastrointestinal disorder of bleeding
Collection of stool:
A. For routine examination-
1. Fresh stool void at anytime of the day may be collected (about 20-30gms) in
a clean and dry container.
2. It should not be allowed to mix with urine.
3. Oil, drugs, barium or bismuth should not be given to the patient prior to the
examination.
4. Stool should be examined within 30 minutes, otherwise trophozoites will be
missed. But when delay is unavoidable or when the parasite is to be
demonstrated later on, it should be preserved by refrigeration at 2°-4°C or
by Formalin.
B. For culture-
1. Collect a suitable amount of stool (10-20gms) in a container or in a wide
mouthed bottle with screw cap which is previously sterilized. For culture the
container must be sterile.
2. Fresh stool void at anytime of the day is the suitable material for culture.
3. In chronic bacillary dysentery cases and in cases where the stool is not
readily available, the rectal swab taken directly by a proctoscope is the
practical method of taking the sample. But it must be ensured that it is taken
from the rectum and not just from the anal orifice.
4. Send the specimen to laboratory without any delay.
***Q-9. Define glycosuria and write down their causes. How can you detect glucose in
urine?
Answer. Glycosuria: The presence of glucose in urine is called glycosuria.
The causes of glycosuria:
1. Diabetes mellitus
2. Hyperactivity of Thyroid, Pituitary and Adrenal cortex, (e.g. thyrotoxicosis,
Cushing’s syndrome, Acromegaly) and injections of these hormones.
3. Severe emotional stress like anger, fear and excitement leads to temporary
glycosuria (due to adrenal secretion by adrenal gland).
4. Severe liver disease or whole organ disease of Pancreas.
5. Infections, Anaesthesia, Asphyxia.
6. Renal glycosuria.
7. Alimentary glycosuria.
8. Central nervous system disorder, e.g. brain injury, stroke.
9. Pregnancy with possible latent diabetes (gastrointestinal disorder).
10. Severe burns
11. Severe sepsis.
Detection of glucose in urine: A. Qualitative tests-

1. Cupper reduction test- a. Benedict’s test
b. Fehling’s test
2. Enzymatic tests- a. Reagent strip test,
b. Clinitest
3. Osazone test
B. Quantitative test-
1. Benedict’s quantitative test
2. Automated urine analysis.
*Q-10. Mention the difference between bacterial, viral and tubercular meningitis.
Answer. The difference between bacterial, viral and tubercular meningitis is given below:
Characteristics Normal Bacterial/Pyogen Tubercular Viral Subarachnoid Tumour
of CSF ic meningitis meningitis meningitis haemorrhage
1. Pressure 60-100 Normal/ Normal/ increased Normal Increased Increased
(mm of H2O) increased
2. Colour Crystal clear Cloudy, Clear/ cloudy Clear Blood mixed/ Clear
purulent. xanthochromic
3. Cell count Lymphocyte Neutrophils Mostly Lymphocyte Red cells Normal
(per cu.mm) s 0-5 often more than lymphocytes upto s upto 2000. mostly
1000- 5000 500
4. Protein 150- 450 Increased Increased Increased Normal Increased
(mg/l) 500-2000 500-3000 500-2000 500-2000
5. Glucose 50- 70 May disappear Decreased 20-30 Normal Normal Normal
(mg/l)
6. Chloride 710 – 750 Normal Less than 600 Normal Normal Normal
(mg/dl)
7. Other Culture- Organisms on Tubercle bacilli in Stain and Centrifuged
changes sterile Gram stain and fibrin clot and culture- No supernatant is
(deposit) culture deposit on ZN stain organism. yellow
and culture; PCR
may identify the
organism
Q- 11. Write the routine examination of CSF with the normal findings.
Answer. Routine examination of CSF with normal findings:
A. Physical examination-
1. Volume: For a normal adult, it is about 150 ml and for neonates it may be 10-60
ml.
2. Appearance and Colour: Clear and colourless.
3. Pressure: Normally for an adult 60- 100 mm of H2O.
4. Specific gravity: Normally 1003 – 1006
5. Blood: Blood normally absent.
6. Clot: Normal CSF does not clot.
1. Protein: Normally 150-450 mg/l.
2. Glucose: Normally 50-70 mg/l.
3. Chlorides: Normally 700-750 mg/dl (120-127 mmol/l).
C. Cytological examination-
1. Cells: Normally 0-5 lymphocytes/cu.mm.
D. Bacteriological- Normally CSF is sterile.
1. Smear: Centrifuged deposits are stained by Gram’s stain and Ziehl-Neelsen’s
stain.
2. Culture and Sensitivity: Chocolate agar media and Lowenstein- Jensen media.
E. Serological- Normally no abnormality is seen.
1. VDRL
2. Kahn test
3. CFT
4. Colloidal gold test- for immunoglobulins.
Q- 12. What is ketonuria and what are the causes? How can you detect ketone bodies in
urine?
Answer. Ketonuria: Ketone bodies are intermediate products for fat metabolism which
comprise of acetone, acetoacetic acid and β- hydroxybutyric acid; and presence of ketone
bodies in urine is called ketonuria.
Causes of ketonuria: A. Physiological-
1. Prolonged starvation
2. Low carbohydrate intake and high fat intake.
B. Pathological-
1. Uncontrolled diabetes mellitus.
2. Pregnancy toxaemia with vomiting.
3. Clinical condition with severe vomiting and toxaemia.
4. Glycogen storage diseases.
5. Febrile state in children.
Detection of ketone bodies in urine: 1. Rothera’s test

2. Gerhardt’s test/ Diacetic acid test
3. Legal’s test
4. Reagent strip test
5. Acetest (Modified Rothera’s test)
Q- 13. How bile salts, bile pigments and urobilinogen can be detected in urine? What is
their clinical significance?
Answer: Detection of bile salts in urine: 1. Hay’s test
2. Strip method.
Detection of bile pigments in urine: 1. Fouchet’s test

2. Foam test
3. Iodine ring test
4. Harrison test
5. Diazo test
6. Gmelin’s test
7. Reagent strip test/ Paper strip test
Detection of urobilinogen in urine: 1. Ehrlich’s test

2. Schlesinger’s test
3. Reagent strip method/ Paper strip test.
Clinical significance of:

A. Bile salts in urine- Bile salts are found in obstructive jaundice.
B. Bile pigments (or, Bilirubin)- Conjugated bilirubin is soluble to water and passes
in the urine in obstructive and some cases of hepatocellular jaundice.
C. Urobilinogen- Excess urobilinogen is found in haemolytic jaundice &
haemolytic anaemia, malaria, congestive heart failure and early hepatocellular damage.
Q- 14. Write down the principle and procedure of Gram’s stain and Ziehl- Neelsen’s stain.
Answer. Gram’s staining (or, Jensen’s modification):
Principle-
When bacteria are stained with a violet stain and then mordanted (it
increases the colouring capacity of the stain) with iodine solution, some of them resist
subsequent treatment with decolourizing agent whereas others do not resist
decolourization. The organisms which resist decolourization are called “Gram-positive”;
and the organisms which are decolourized are called “Gram-negative”. The Gram-negative
organisms are then coloured with a red counter stain to differentiate from the Gram-
positive organisms.
Reagents-
1. Stain: Crystal violet or methyl violet or gentian violet- 0.5% solution in
distilled water.
2. Lugol’s iodine solution:
Iodine ................................... 1 gm
Potassium iodide ................. 2 gm
Distilled water .................... 100 ml
3. Acetone or alcohol
4. Counter stain: a. Neutral red-
1 gm neutral red
2 ml 1% Acetic acid
Distilled water to make 500 ml.
b. Safranin-
1.7 gm safranin
50 ml alcohol
Distilled water to make 500 ml.
c. Diluted Carbol-fuchsin-
1:10 dilution of strong carbol-fuchsin.
Method/Procedure-
1. Preparation and fixation of the film: The slide should be clean and free from grease.
The material, if solid, is emulsified in a loop of clean water on the slide with a
sterile wire loop. A thin, uniform film is made with the loop. The film is dried in the
air and then fixed by passing the slide 3-4 times slowly through the flame. When
the slide is just hot to be borne on the back of the hand, the fixation is complete.
The slide is cooled.
2. Staining: Place the slide on the rack and flood with crystal violet or gentian violet
or methyl violet stain and kept for one minute.
3. Mordanting: The stain is drained off and washed with Lugol’s iodine solution or
Grams iodine solution. More iodine solution is poured on the film and kept for 2
minutes.
4. Decolourization: The iodine solution is washed off with alcohol or acetone and then
washed with water. The alternate washings with alcohol and water are repeated till
the colour ceases to come out of the film. (Acetone does this more quickly than
alcohol, so care should be taken not to use acetone for a longer period. Serous and
mucoid materials are more difficult to decolourize than saline suspensions and
required a longer exposure to the decolourizing agent).
5. Counter staining: It is counter stained with 1 in 10 diluted carbol-fuchsin solution
for 20 seconds or with neutral red for 2 minutes or with safranin for 1 minute.
6. Wash with water and dried in air or dried by blotting it with filter paper.
7. Examine under oil immersion of the microscope.
Results-
Because the Gram-positive organisms retain the crystal violet after
decolourization, they appear dark blue in colour. The Gram-negative organisms are
decolourized and take up the colour of the counter stain and appear pink in colour.
Acid Fast Staining or AFB staining or Ziehl Neelsen’s Stain:

Principle-
When bacteria are stained with hot carbol fuchsin, some of them resist
decolourization by subsequent treatment with mineral acids, whereas others do not resist
decolourization. The organisms which resist decolourization is called Acid Fast Bacteria,
and the organisms which do not resist decolourization are called non-acid fast bacteria.
Reagents-
1. Carbol-fuchsin [Ziehl Neelsen’s (strong) Carbol fuchsin]:
Basic fuchsin .......................................... 10 gm
Absolute Ethanol ................................... 100ml
5% solution of Phenol in water ............ 1000 ml
2. Decolourizing agent:
20 % Sulphuric acid (H2SO4) for Mycobacterium tuberculosis
05% Sulphuric acid (H2SO4) for Mycobacterium leprae, or
Acid alcohol 3% Hydrochloric acid (HCl) in 95% alcohol,
(or, 01% Sulphuric acid (H2SO4) for Actinomyces)
3. Counter stain: a. Luffler’s methylene blue, or Malachite green, or Picric
acid.
Methods/Procedure-
1. A film is made from the material. Dried in air and fixed by passing through
flame. The film should be 10-20 mm in diameter.
2. Flood the whole slide with strong carbol fuchsin and heat gently underneath
the slide until steam is seen rising from the slide and then kept for 5
minutes. (Do not overheat; avoid boiling over of the stain). or, Carbol
fuchsin is heated in a test tube till fumes appear. Then the slide is covered
the fuming carbol fuchsin and kept for 5 minutes.
3. The slide is washed with water to remove the excess stain.
4. a. The film is washed with 20% sulphuric acid to see whether it is acid fast.
Washings with the sulphuric acid and water are repeated until the wash is
faint pink in colour.
b. To find out whether the organism is also alcohol fast, the film is washed
with 95% alcohol for 2 minutes. Instead of employing sulphuric acid and
alcohol in two steps, the acid- alcohol (3% HCl in 95% alcohol) may be
used to find out acid and alcohol fast at the same step.
5. The slide is rinsed in water.

6. Counter stain with methylene blue for 2 minutes or with diluted malachite
green for 1 minute, or with picric acid.
7. Wash with water and dried in air. (Do not blot).
8. Examine under the oil immersion of the microscope.
Result-
The tubercle bacilli resist decolourizing by acid and alcohol (i.e. it is both
acid and alcohol fast), it remains bright red while all the other organisms
and material will take up the colour of the counter stain.
Q- 15. Write short notes on: *** i. OBT.

ii. OGTT.
*** iii. Bence Jones protein.
iv. CPK
v. Body fluids
vi. Haematuria and Haemoglobinuria.
vii. Urinary casts and crystals
viii. Total count of spermatozoa.
vii. Characteristics of common ova and cysts
Answer.
*** i. OBT (Occult Blood Test): The test that is done to detect the blood in stool
which is not discernable to naked eye is known as occult blood test (OBT).
Methods-
a. Benzidine test
b. Orthotoluidine test
c. Guaiac test
d. Alvarez & Wright’s Modification of Gregersen Test
e. Aminophenazone test
f. Haema-check slide test/ Reagent paper strip test
Causes of occult blood in stool-

a. Peptic ulcer
b. Acute and chronic inflammatory lesions, like amoebic dysentery, bacillary
dysentery, ulcerative colitis etc.
c. Oesophageal varices.
d. Malignant tumours of the Gastro-intestinal tract, pancreas, ampulla of Vater etc.
e. Ulcer in the Meckel’s diverticulum.
f. Diseases due to bleeding diathesis, e.g. haemophilia, purpura.
g. Hookworm disease
ii. OGTT (Oral glucose tolerance test)/Glucose tolerance test (GTT): Glucose
tolerance test (GTT) or Oral glucose tolerance test (OGTT) determines the ability of an
individual to utilize a given quantity of glucose taken orally.
Preparation of the individual for the test-
The individual should be on a normal diet for 3 days preceding to the day of the
test. He is to fast overnight for about 8-12 hours. Water is allowed. He should be free
from other illness and effects of trauma and corticosteroid, diuretics, phenothiazines,
anti-depressants as these decrease glucose tolerance test.
Test procedure-
The patient should remain seated and refrain from food and smoking during the test.
The fasting blood and urine samples are collected. 75 gram glucose in 250-300 ml water
(can also be given with lemon water) is given orally. And then half hourly blood and urine
samples are taken for 2 hours. All the samples are tested for and the glucose tolerance
curve is plotted with the time as abscissa and blood glucose values as ordinates. Urine
glucose values are recorded below the corresponding blood glucose values under the
abscissa.
Normal Glucose Tolerance Test Curve-
Fasting After taking 75gm glucose by mouth Corresponding Urine
½ hour 1 hour 1 ½ hour 2 hours sugar
4.0 5.5 4.6 4.4 4.0 Absent in all samples
mmol/l mmol/l mmol/l mmol/l mmol/l
Indication of glucose tolerance test-

1. Persistent glycosuria, hyperglycaemia and syneglycosuria.
2. To determine the severity of diabetic condition.
3. In individuals with quickly developing obesity.
4. In the event of fasting sugar more than 120 mg%.
5. In persons whose parents are diabetic.
6. In case of unexplained periodic weakness or convulsive seizures suggestive of
hyperinsulinism.
*** iii. Bence Jones protein: Bence Jones proteins represent either the κ (kappa) or
λ (lambda) light chains of the immunoglobulin formed in some lymphoproliferative
disorders. It was first demonstrated by H. Bence Jones in 1847.
Properties of Bence Jones proteins-
Bence Jones protein develops at 40° -60°C and redissolves on 100°C. On cooling
again, the precipitate reappears at 40° -60°C. While other urinary proteins usually appear at
60°C and do not redissolve on boiling.
Detection of Bence Jones protein in urine-
Filter the urine, if cloudy. If alkaline, make it acidic by with 5% acetic acid. Place 5
ml of urine in a test. Place the test tube in the water bath and raise the temperature
gradually. Bence Jones protein begins to appear at 40° -60°C. Heating is continued and the
Bence Jones protein redissolves on boiling. Filter the boiling urine and allowed to cool.
The reprecipitation of the filtrate urine at 40° -60°C indicates presence of Bence Jones
protein.
Bence Jones Protein found in-
a. Multiple myeloma.
b. Malignant lymphoma.
c. Chronic myeloid leukaemia.
d. Osteosarcoma.
e. Osteomalacia.
f. Generalized carcinomatosis.
iv. CPK (Creatinine Phosphokinase)/ Creatinine Kinase: It is an enzyme found in

high concentration in muscle, heart and brain. It consists of 3 major dimeric isoenzymes-
a. CK1 or CK-BB: Found in the brain.
b. CK2 or CK-MB: Found in the heart.
c. CK3 or CK-MM: Found in the skeletal muscle.
Normal values-
Male: 30-200 U/L
Female: 30-150 U/L
Infants: 2-3 times adult values.
Normal values of isoenzymes-
a. CK1 or CK-BB: 00%
b. CK2 or CK-MB: 00-06%
c. CK3 or CK-MM: 96-100%
Clinical significance- It is done in case of suspected myocardial infarction, muscle
diseases and hypothyroidism where CK level is considerably raised.
v. Body fluids: Water of the body together with its dissolved solute is called body
fluid. So, body fluid is an aqueous solution containing electrolytes and non-
electrolytes and consists of intracellular and extracellular components.
Types-
1. Intracellular fluid (ICF): The fluid inside the cells of the body is known as
intracellular fluid. It is about 40% of the total body weight and is about the 60 -
70% of the total body fluid.
2. Extracellular fluid (ECF): The fluid in the spaces out side the cells of the body
known as extracellular body fluid. It is about the 20% of the total body weight
and is about 30-40 % of the total body fluid.
Total Body Fluid (42 L)
Intracellular fluid (28 L) Extracellular fluid (14 L)
Interstitial fluid (11 L) Plasma (3 L) Trancellular fluid (1-2 L)
vi. Haematuria and Haemoglobinuria: Presence of red cells in urine is called

haematuria and passage of haemoglobin in urine is called haemoglobinuria.
Methods of detection of haematuria/ detection of blood in urine-
1. Microscopic examination of urine for RBC.
2. Chemical examination for haemoglobin:
a. Orthotolidine test
a. Benzidine test.
Causes of haematuria-
A. Pre-renal:
a. Collagen disease
b. Filariasis (lymphatic obstruction)
c. Leukaemia
d. Purpuric diseases
B. Renal:
a. Polycystic kidney
b. Acute glomerulonephritis (AGN)
c. Focal or embolic glomerulonephritis
d. Renal tuberculosis
e. Malignant hypertension
f. Nephrolithiasis
g. Renal tumours
C. Post-renal:
a. Traumatic after catheterization.
b. Stone in the urinary bladder
c. Tumours in the urinary bladder
d. Prostitis and urethritis
e. Benign prostatic hypertrophy (BPH)
f. Cancer of the prostate
Causes of haemoglobinuria-
a. Incompatible blood transfusion
b. Black water fever in malignant malaria
c. Paroxysmal nocturnal haemoglobinuria
d. Paroxysmal cold haemoglobinuria
e. Streptococcal septicaemia
f. Gas gangrene
g. Strenuous exercise
vii. Casts and crystals (unorganized elements) of urine:

Urinary Casts-
These are long, narrow, cylindrical structure with parallel sides and
rounded ends and are formed by molding of hollow structures on themselves. They
are of different size and shapes.
Various casts found in urine are-

1. Cellular cast:
a. Epithelial cast- found in AGN, and degeneration of renal tubule.
b. RBC cast- found in Acute Glomerulonephritis (AGN)
c. WBC cast- found in inflammation of renal tubule.
2. Hyaline cast: found in glomerulonephritis and hypertension.
3. Granular cast: found in inflammation and degeneration of renal tubule.
4. Fatty cast: found in marked degeneration of kidney.
5. Colloid cast: found in nephrosis.
6. Amyloid cast: found in amyloidosis of kidney.
Crystals
Crystals are the unorganized deposits of urine found mostly in various
disease conditions. Commonly found crystals are-
A. Crystals of Acidic urine:
1. Amorphous urate crystal
2. Uric acid crystals
3. Cholesterol crystals.
4. Calcium oxalate crystals
5. Sodium urate crystals
6. Cystine crystals
7. Fat droplets
8. Leucine spheres
9. Tyrosine needles
B. Crystals of Alkaline urine
1. Amorphous phosphate
2. Calcium carbonate
3. Calcium phosphate
4. Ammonium urate.
v. Total count of spermatozoa:

Requirements-
1. Haemocytometer: It contains –
a. RBC pipette
b. WBC pipette
c. Improved Neubauer counting Chamber.
d. A special coverslip
2. Diluting fluid:
Sodium bicarbonate - 5 gm
Formalin neutral - 1 ml
Distilled water - 100 ml
3. Sample
Procedure-
1. Mix the specimen thoroughly by shaking the container tube gently.
2. Draw the semen upto 0.5 mark of a white cell diluting pipette and then the
diluent upto the 11 mark. Mix well by rotatory and side to side movements.
3. Fill the counting chamber (Neubauer ruling) with the diluted seminal fluid
and leave on the bench for 2 minutes to settle down the immobilized sperm.
4. Count the number of sperm in four large corner squares covering 4 sq.mm
area under high power lens of the microscope. While counting only consider
those spermatozoa which are complete, i.e. with a head, a neck and a tail.
Calculations-
Total spermatozoa per ml = Sperm count × 50,000
ix. Characteristics of common ova and cysts:
A. Commonly found ova-

a. Ova of Ankylostoma duodenale/ Necator americanus (Hookworm)-
1. Shape: Oval or elliptical
2. Size: 65µm × 40µm
3. Colour: Colourless
4. Egg shell: Transparent and thin
5. Not bile stained and floats on saturated salt solution.
b. Ova of Ascaris lumbricoides (Round worm)-

I. Fertilized ova of Ascaris lumbricoides
1. Shape: Oval or round
3. Colour: Yellow brown bile stained.
4. Egg shell: Smooth, translucent, mamilated.
5. Ovum: Unsegmented ova
6. Albuminous coat- Present.
I. Unfertilized ova of Ascaris lumbricoides

1. Shape: Elongated
3. Colour: Brown
4. Egg shell: Very thin, transparent, hyaline membrane and not mamilated.
5. Ovum: Atrophoid ova with disorganized retractile granules.
6. Albuminous coat- Absent
7. Does not float on saturated salt solution.
c. Ova of Trichuris trichiura (Whipworm)-

1. Shape: Barrel shape with mucus plug at each end.
3. Colour: Brown and bile stained.
4. Egg shell: Thick, double shell, outer one is bile stained.
5. Floats on saturated salt solution.
d. Ova of Enterobius vermicularis (Pinworm)-

1. Shape: Plano-convex
3. Colour: Colourless
4. Egg shell: Transparent and has a coiled up larva within it.
5. Not bile stained and floats on saturated salt solution.
e. Ova of Taenia saginata/ T. solium/ Echinococcus granulosus-

1. Shape: Round
2. Size: 35-45µm in diameter
3. Colour: Brown in colour (bile stained)
4. Egg shell: Thin, transparent an contains embryo with six hooklets.
5. Floats on saturated salt solution.
B. Commonly found cysts-

a. Entamoeba histolytica:
1. Trophozoite or vegetative form of Entamoeba histolytica-
i. Size: 10 - 40 µm
ii. Shape: Not fixed
iii. Movement: Motile
iv. Nucleus: Spherical in shape having a nuclear membrane and a central
karyosome.
v. Cytoplasm:
-Ectoplasm and
- Endoplasm, which may contain RBC and other particle.
2. Cystic form of Entamoeba histolytica-
i. Shape: Round
ii. Size: 5-20 µm in diameter.

iii. Nucleus: 2-4.
b. Giardia:
1. Cystic form of Giardia lamblia-
i. Size: 8-14 µm in length and 5-10 µm in width
ii. Shape: Oval
iii. Nucleus: 2-4
iv. Colour: Colourless
2. Trophozoite of Giardia lamblia-
i. Shape: Pear
ii. Size: 15 µm × 9 µm
iii. Nucleus: 2 with large central karyosome.
iv. Flagella: 4 pairs or 8 in numbers.
v. Colour: Colourless.
Structures seen in saline preparations-

1. Ova of Ankylostoma duodenale
2. Ova of Enterobius vermicularis
3. Ova of Necator americanus
Structures seen in iodine preparations-
1. Ova of Ascaris lumbricoides and Trichuris trichiura.
2. Cysts of Entamoeba histolytica, Entamoeba coli, Giardia lamblia, G. intestinalis.
Ova those float on in Saturated Salt Solution

1. Fertilized ova of Ascaris lumbricoides
2. Ova of Ankylostoma duodenale
3. Ova of Trichuris trichiura
4. Ova of Enterobius vermicularis
5. Ova of Hymenolepis nana.
Ova those do not float on Saturated Salt Solution-

1. Ova of Taenia
2. Ova of Flukes
3. Ova of Diphylobothrium latum (D. latum)
4. Unfertilized ova of Ascaris lumbricoides.
“We can never be happy, if we are not satisfied with our selves. And
the first step to happiness is to shun the ego.” Monir Ahmed
Haematology
***Q- 1. Define erythropoiesis. What are the sites and stages of erythropoiesis? Name the
factors affecting erythropoiesis.
Answer. Erythropoiesis: The process of formation of erythrocytes under normal
physiological condition is known as erythropoiesis.
Sites of erythropoiesis:
1. Mesoblastic erythropoiesis- is the production of primitive, nucleated erythrocytes in
the mesoderm of the yolk sac in the early few weeks of embryonic life.
2. Hepatic erythropoiesis- is the production of primitive erythrocytes mainly in the
liver during the middle trimester of gestation. In this period a reasonable numbers
of erythrocytes are also produced by the spleen and lymph nodes.
3. Myeloid erythropoiesis- is the production of erythrocytes by the bone marrow
during the last trimester of gestation and after birth.
Stages of erythropoiesis:
1. Proerythroblast/ Pronormoblast
2. Basophil erythroblast/ Early normoblast
3. Polychromatophil erythroblast/ Intermediate normoblast
4. Orthochromatic erythroblast/ Late normoblast
5. Reticulocyte
6. Mature erythrocyte
Factors affecting erythropoiesis:

A. General factors-
1. Diet
2. Hypoxia
3. Erythropoietin
4. Endocrine hormones: a. Pituitary hormone- ACTH (Adrenocorticotropic hormone)
b. Thyroid hormone- Thyroxin (T4)
B. Haemoglobinization factor-
1. Iron (Fe)
2. Cobalt (Co)
3. Calcium (Ca)
4. Bile salts
C. Maturation factors-
1. Vitamin B12
2. Folic acid
3. Intrinsic factor of Castle
**Q- 2. Define and classify anticoagulants with examples. What are the anticoagulants
commonly used in the laboratory? Why EDTA is best?
Answer. Anticoagulants: Anticoagulants are substances which prevent clotting of blood.
Classification of anticoagulants:
A. General classification-
1. Natural anticoagulants-
a. Heparin
b.Antithrombin III (Heparin co-factor II)
2. Artificial anticoagulants (Commonly used in the laboratory) -
a. Heparin
b. Ethylenediamine tetra-acetic acid (EDTA)
c. Oxalated compounds:
i. Single oxalate or Potassium oxalate
ii. Double oxalate or Paul Heller’s mixture (Ammonium oxalate+ Potassium
oxalate).
d. Tri-sodium citrate
e. Acid citrate dextrose
f. Citrate phosphate dextrose
g. Alsever’s solution
h. Sodium fluoride
B. According to mode of action-

1. Acting in vitro (outside the body), e.g. EDTA, heparin, Na-citrate, potassium
oxalate etc.
2. Acting in vivo (inside the body), e.g. Dicoumarol, warfarin, phenindione etc.
3. Acting vitro and vivo, e.g. heparin, dextran sulphate.
EDTA is best or ideal, because-

1. Morphology of RBC and WBC can easily be identified.
2. It prevents the clumping of the platelets, thus making the platelet count more
accurate.
3. Bone marrow study can be done by using 2% solution of EDTA.
4. It does not interfere in the most of the bio-chemical tests.
*Q-3. What are the criteria of a good film? Write down the steps of preparation of a blood
film.
Answer. The criteria of a good film:
1. It should be uniformly thick but not too thick.
2. The margin should be even and should not touch the side of the slide.
3. The film should be tongue shaped having three parts- head, body and tail with a
length about 3 -4 cm (occupying the middle two-third of the slide).
4. There should be a single tail which is not too long and should be gradually thin
without any serrated ends.
5. The film should end near the centre of the slide and not extending upto the edge
of the slide.
6. The cells should be uniformly distributed especially white cells.
7. There should be some overlapping of red cells near the head of the slide but are
separated from each other near the tail.
8. On the surface of the film, there should be no ridges, weaves and holes.
9. The film should be properly stained to identify the cells morphology.
The preparation of a blood film:

1. Put one drop of blood over one end of the surface of a clean and grease free
slide from a finger prick or from a needle of a syringe.
2. Keep the slide on a flat surface so that the blood drop is on the right hand side.
3. Fix the slide with the left hand by holding the left side between thumb and
finger.
4. With the right hand, place the spreading edge of the spreader on the slide just in
front of the blood drop at an angle of about 45° on the right.
5. Glide back the spreader till it touches the drop of the blood. The blood will
immediately spread to the whole margin of the spreader.
6. Keeping the spreader in same angle, give a quick even push with a uniform
force towards the other end of the slide. This will bring forward the blood and a
tongue shaped smear will be produced.
7. Allow the film to air dry with the slide in a horizontal position.
***Q-4. Define MCH, MCV and MCHC with their normal values.
Answer. Red cell indices/ absolute values: a. Mean corpuscular volume (MCV)
b. Mean corpuscular haemoglobin (MCH)
c. Mean corpuscular haemoglobin concentration
(MCHC).
Mean corpuscular volume (MCV): This is the mean or average volume of a single
red cell expressed in femtoliter.
PCV l/l
Calculation- MCV = femtoliter
RBC count per liter
PCV % × 10
= femtoliter
RBC/cu. mm
Normal value- 76 – 96 fl (1 fl= 10-15 L)

Mean corpuscular haemoglobin (MCH): It is the average amount of haemoglobin

present in each red cell and expressed in picogram.
Hb concentration per liter
Calculation- MCH = picogram
RBC count per liter
Hb gm/dl × 10
= picogram
RBC / cu. mm ×1000 × 1000
Normal value- 27 – 32 pg (1 pg= 10-12 gm)
Mean corpuscular haemoglobin concentration (MCHC): It is the amount of
haemoglobin present per unit volume of the red cells and expressed as gm/dl.
Hb gm/dl Hb gm/dl
Calculation- MCHC = = × 100
PCV l/l PCV in percentage
Normal value- 31 – 35 gm/dl.
**Q-5. What are the causes of iron deficiency anaemia? Give the laboratory diagnosis of
iron deficiency anaemia.
Answer. Iron deficiency anaemia: It develops when the supply of iron to the bone marrow
is insufficient for the requirement of Hb synthesis.
Causes of iron deficiency anaemia-
A. Increased physiological demands:
1. Children during the period of growth
2. Premenopausal women
3. Pregnancy
B. Pathological blood loss:
1. Peptic ulcer
2. Haemorrhoids (Piles)
3. Hiatus hernia
4. Carcinoma of stomach
5. Carcinoma of colon
6. Oesophageal varices (when rupture, there is severe bleeding)
8. Hookworm infestation
9. Urinary tract infection and haematuria
10. Menorrhagia in female
C. Malabsorption/ Impaired absorption:
1. Coeliac disease
2. Post-gastrectomy
3. Tropical sprue
4. Whipples diseases
5. Atrophic gastritis
D. Nutritional deficiency:
1. Inadequate intake or improper feeding.
Laboratory diagnosis of iron deficiency anaemia:

A. Blood picture-
1. Haemoglobin- variably reduced.
2. Blood film- Hypochromia, microcytosis, anisocytosis, poikilocytosis. In severe
cases target, elliptical, oval and pencil cells.
3. Red cells- Total count is normal or reduced and osmotic fragility reduced.
4. Haematocrit- Reduced.
5. Red cell indices- MCV is reduced (microcytic), MCHC is reduced
(hypochromic) and MCH is reduced.
6. Reticulocyte count- Normal.
B. Iron Profile-
1. Serum iron is reduced- less than 10 µmol/l.
2. Serum ferritin is reduced- less than 12µg/l.
3. Total iron binding capacity (TIBC) is increased- 80 µmol/l or more.
4. Percentage saturation of iron binding protein is decreased below 16%.

C. Bone marrow-
1. Erythroid hyperplasia: Micronormo-blastic erythropoiesis.
D. Further investigations-
1. Stool examination:
a. Ova of hookworm, and
b. Occult blood test.
2. Urine examination for red cells.
3. Other investigation depending on clinical findings, e.g. Chest X-ray for
haemoptysis,
***Q-6. Define and classify leukaemia. Give the laboratory diagnosis of acute myeloid
leukaemia (AML) and chronic myeloid leukaemia (CML) or, Give the bone marrow
findings of AML and CML.
Answer. Leukaemia: The leukaemias are diseases of unknown aetiology characterized by
uncontrolled abnormal and widespread proliferation of the leukocytic cells of the body
which infiltrate the bone marrow and other body tissues usually associated with but not
invariably presence of immature white cells in the peripheral blood. It can also be defined
as, “a malignant state of haemopoietic tissue characterized by widespread proliferation of
leucopoietic cells in bone marrow with or without appearance of abnormal premature
leucocytes in peripheral blood.”
Classification of Leukaemia:
A. Classification on Clinical course-
1. Acute leukaemia
2. Chronic leukaemia
B. Morphological classification- based on predominant leukaemic cells.
1. Myeloid leukaemia
2. Lymphoid leukaemia
C. Combined classification (Clinical and Morphological)-
1. Myeloid leukaemias
a. Acute myeloblastic leukaemia/ Acute myeloid leukaemia.
b. Chronic myelocytic leukaemia/ Chronic myeloid leukaemia.
2. Lymphoid leukaemias
a. Acute lymphoblastic leukaemia.
b. Chronic lymphocytic leukaemia.
Laboratory diagnosis of Acute myeloblastic leukaemia (AML):

A. Blood picture-
1. Haemoglobin: Reduced (3 – 9gm/dl).
2. PCV: Reduced.
3. Peripheral blood film:
a. RBC- Predominant cells are normocytic and normochromic. Some are
microcytes with anisocytosis and poikilocytosis. Mild polychromasia. A few
nucleated red cells may appear.
b. WBC-
i. Count: 20,000-100,000/ cu. mm of blood.
ii. Immature blast cells: 80-90%.
iii. Myeloblast, monoblast, lymphoblast are difficult to differentiate.
iv. Auer bodies present in myeloblast.
v. Smear cells absent.
c. Platelet- Thrombocytopenia.
d. Reticulocytes- upto 05%.
B. Bone marrow-
1. Cellularity: Hypercellular.
2. M/E ratio: Increased.
3. Erythropoiesis: Decreased.
4. Leukopoiesis: Increased, immature blast cells are 30-90%.
5. Megakaryopoiesis: Reduced.
C. Special investigations-
1. Serum lysozyme (muramidase).
2. Cell surface markers.
3. Bone marrow: Chromosomal abnormality upto 75%.
Laboratory diagnosis of chronic myeloblastic leukaemia (CML)/Chronic

granulocytic leukaemia (CGL):
A. Blood picture-
1. Haemoglobin: Variably reduced (3 – 9gm/dl).
2. PCV: Reduced.
3. ESR: Moderate to high rise.
4. Peripheral blood film:
a. RBC- Normocytic normochromic anaemia. Polychromasia and basophilic
stripling. Normoblasts may be present.
b. WBC-
i. Count: 95,000-600,000/ cu. mm of blood.
ii. Myelocytes are predominant cells.
iii. Myeloblast (<10%), myelocytes (20-30%), metamyelocytes (15-20%)
and segmented neutrophils are (30-60%), and rest other leukocytes.
c. Platelet- Increased (162-2000 × 109/L). At a later stage reduced. Atypical
large platelets are common.
d. Reticulocyte count- Moderate reticulocytosis.
B. Bone marrow-
1. Cellularity: Hypercellular.
2. M/E ratio: Increased.
3. Erythropoiesis: Depressed.
4. Leukopoiesis: Active. Majority of the cells are of the myeloid series, predominantly
myelocytes.
5. Megakaryopoiesis: Increased
C. Special investigations-
1. Chromosome study (Bone and Marrow): Presence of Philadelphia (Ph)
Chromosome is found in about 90% cases.
2. Serum vitamin B12 level is high.
3. Serum lactate dehydogenase level is high.
4. Alkaline phosphatase in neutrophils- Low.
5. Serum uric acid- Increased.
**Q-7. Give the uses of Improve Neubauer Counting Chamber, Wintrobe tube, RBC &
WBC pipette.
Answer. Uses of Improved Neubauer Counting Chamber: It is used for-
1. Total count of RBC
2. Total count of WBC
3. Total count of cells in different body fluids, e.g. CSF, synovial fluid etc.
4. Total count of Spermatozoa in seminal fluid.
Uses of Wintrobe tube: It is used in the-

1. Determination of ESR
2. Determination of PCV/Haematocrit (Hct)
3. Buffy coat preparation
Uses of RBC pipette: It is used in the dilution for the determination of –

1. Total count of RBC
2. Total count of WBC in case of leukaemia where the count is very high.
3. Total count of Platelets.
Uses of WBC pipette: It is used in the dilution for the determination of-
1. Total count of WBC
2. Total count of cells in different body fluids, e.g. CSF, synovial fluid etc.
4. Total count of platelets when platelet counts are low, e.g. Dengue fever.
***Q-8. Give the total count, differential count and absolute count of WBC. Draw and
label WBCs.
Answer. Normal Total count of WBC: 4000-11000/cu.mm. of blood.
Absolute count and Differential count of WBC-
◊ Granulocytes-
⇒ Neutrophils (Polymorphs.) : 2000-7500/µl of blood. 40-75%
⇒ Eosinophils : 100-400/µl of blood. 1-4%
⇒ Basophils : 0-100/µl of blood. 0-1%
◊ Agranulocytes-
⇒ Lymphocytes : 2000-4500/µl of blood. 20-45%
⇒ Monocytes : 200-800/µl of blood. 2-8%
Figure- White Blood cells
Q- 9. Define PCV. Mention the causes of high and low PCV.

Answer. PCV (Packed cell volume): PCV or Haematocrit is the volume of red cells in
relation to that of whole blood. PVC may be define as, “the volume of red blood cells
expressed as percentage of the total volume of blood”. Actually, it is the ratio of packed red
cells and whole blood volume expressed in percentage or volume per volume.
Causes of High PCV:
1. Physiological-
a. High altitude
b. New born and infants
c. Excessive sweating in hot climatic condition (due to haemoconcentration).
2. Pathological-
a. Polycythaemia
b. Conditions in which there is haemoconcentration, e.g.
i. Severe vomiting and diarrhoea
ii. Shock
iii. Burn
Causes of Low PCV:

1. Physiological-
a. Pregnancy
b. Excessive water intake
c. Infusion of saline
2. Pathological-
a. Anaemia
b. Leukaemia, lymphoma, Hodgkin’s disease, myeloproliferative disorders.
c. Adrenal insufficiency
d. Acute and chronic blood loss
e. Haemolytic reaction
f. Hyperaldosteronism
Q- 10. Name the methods of Haemoglobin estimation. Give the advantages and
disadvantages of Acid Hematin Method.
Answer. Methods of estimation of haemoglobin:
1. Visual methods-
a. Sahli's method/Acid haematin method/ Sahli's Acid haematin method
b. Paper strip comparator method
2. Colorimetric methods-
a. Cyanomethaemoglobin method,
b. Carboxy-haemoglobin method
c. Oxyhaemoglobin method
d. Alkaline haematin method
3. Autoanalyzer methods-
a. Electronic counter
b. Direct reading electronic haemoglobinometer
4. Others-
a. Measurement of O2 carrying capacity
b. Measurement of Iron content of Hb
c. Specific gravity method
Advantages of Acid Haematin method:

1. Simple and easy to perform which can be done at the bedside.
2. Reagents and apparatus are cheap.
3. Results can be given in percentage and in gram/dl at the same time.
Disadvantages of Acid Haematin method:
1. There can be visual error (5-10%).
2. Carboxyhaemoglobin, methaemoglobin, sulfhaemoglobins can not be converted.
3. Non-haemoglobin substances (e.g. protein, lipids) in plasma and cell stroma may
influence the colour of blood diluted with acid.
4. Comparator may fade over the years.
5. Colour of acid haematin also fades quickly.
Q- 11. What is leukaemoid reaction? Give the differences between leukaemia and
leukaemoid reaction.
Answer. Leukaemoid reaction: It is used to describe the conditions in which peripheral
blood picture resembles that of leukaemia in a patient who does not have leukaemia usually
with no leukaemic changes in the bone marrow.
Characters Leukaemoid reactions Leukaemia
1. Clinical Clinical feature of the underlying Splenomegaly, Lymph node
Features cause is obvious. enlargement, Haemorrhage more
marked.
2. Blood examinations
a. Total white Mostly below 100,000/mm3 Can exceed 100,000/mm3
cell count
b. Immature cells Less than 10% Usually numerous
c. White cell Toxic changes may be seen. Cells usually atypical as well as
morphology immature. Toxic changes are
uncommon.
d. Anaemia Slight or absent Usually progressive
e. Platelets Normal or increased Decreased except in CGL
f. Nucleated red Frequent in leukoerythroblastic Infrequent and seldom in
cells anaemia number.
3. Bone marrow Mild leucoblastic hyperplasia may Hyperplasia. Large number of
be present. Not like leukaemia. immature cells. Leukaemic
picture.
4. Autopsy
a. Infiltration of Absent Present
organs and
tissues
b. NAP score Increased Normal or decreased.
**Q-12. Define and classify anaemia. What are the causes of anaemia?
Answer. Definition of anaemia: Two important definitions of anaemia are cited below-
a. Anaemia is a clinical condition characterized by a pale colouration of skin and
mucus membrane as a result of qualitative and quantitative deficiency of
haemoglobin below normal range for an individual in respect of age and sex.
b. Anaemia may be defined as reduction in the concentration of haemoglobin in
the peripheral blood below normal for age and sex of the patient.
In adult male haemoglobin less than 13 gm/dl (70%) and in female less than 11.5 gm/dl
(60%) is labelled as anaemia. When haemoglobin is low upto 11 gm/dl it is labelled as mild
anaemia, value of 9-11 gm/dl as moderate anaemia, and less than 9 gm/dl as severe
anaemia.
♦ Classification of anaemia:
I. Morphological classification-
This is based on mainly MCV (Mean Corpuscular Volume), MCHC (Mean
Corpuscular Haemoglobin Concentration), and MCH (Mean Corpuscular Haemoglobin).
1. Microcytic Hypochromic anaemia: MCV, MCHC and MCH are below normal, e.g.
iron deficiency anaemia (most common), thalassaemia, anaemia of chronic diseases and
sideroblastic anaemia.
2. Macrocytic anaemia: MCV is above normal, and MCHC is normal. It occurs in
megaloblastic anaemia and macro-normoblastic anaemia.
3. Normocytic Normochromic anaemia: MCV, MCHC and MCH are within normal
range, e.g. acute blood loss, most cases of anemia due to depression of erythropoiesis and
haemolytic anaemia.
II. Aetiological classification/ Causes of Anaemia-
This classification is based on pathophysiology and cause.
A. Impaired red cell formation / production (Dyshaemopoietic anaemia):
1. Deficiency of essential nutrients (or, Deficiency anaemia) e.g. Iron deficiency
anaemia, Megaloblastic anaemia/ Vitamin B12 deficiency anaemia.
2. Depression of erythropoiesis/ Disturbance of bone marrow function not due to
deficiency of substance essential for the erythropoiesis, e.g. Aplastic anaemia, Sideroblastic
anaemia.
B. Increased destruction of red cell (Haemolytic anemia):
1. Intracorpuscular effect/ Intrinsic abnormality.
2. Extracorpuscular effect/ Extrinsic abnormality.
C. Blood loss (Haemorrhagic anaemia):
1. Acute post-haemorrhagic anaemia-
Loss of large volume of blood over a short period, e.g. surgical blood loss, accidental
blood loss.
2. Chronic post-haemorrhagic anaemia-
e.g. hookworm infection, bleeding peptic ulcer, menorrhagia.
*Q-13. What is ESR? Give the normal values of ESR. Mention the causes of high and low
ESR. Describe the procedure of estimation of ESR by Westergren method.
Answer. ESR: When the blood is mixed with a suitable anticoagulant and is allowed to
stand vertically, red cells settle down to the bottom. The rate at which this sedimentation of
red cells takes place is known as Erythrocyte Sedimentation Rate (ESR).
• Normal values or range of ESR:
Method Normal range or value
◊ Westergren method Men : 0-09 mm in 1st hour.
Women : 0-20 mm in 1st hour.
◊ Wintrobe method Men : 0-10 mm in 1st hour.
Women : 0-20 mm in 1st hour.
Causes of High ESR:

a. Physiological Causes-
1. Pregnancy and puerperium.
2. After vaccinations and inoculations.
3. In old age (above 60 years) ESR increases.
4. In females ESR is slightly higher.
b. Pathological Causes-
1. Infections, e.g. tuberculosis and other chronic infections.
2. Fever.
3. Connective tissue diseases and collagen diseases, e.g. acute rheumatic fever, SLE.
4. Malignant diseases/Neoplasm.
5. Internal haemorrhage.
6. Anaemia.
7. Paraproteinaemia, e.g. Multiple myeloma, macroglobulinaemia.
8. Inflammatory diseases, e.g. acute pelvic inflammatory disease.
9. Hypothyroidism and hyperthyroidism.
10. Acute myocardial infarction.
Causes of Low ESR/ ESR decreases in-

1. Polycythaemia
2. Sickle cell anaemia.
4. Newborn infants
5. Spherocytosis
6. Hypofibrinogenaemia.
Estimation of ESR by Westergren method:

⇒ Materials required-
1. Westergren tube with stand, 2. ESR fluid (3.8% Trisodium/ Sodium citrate),
3. Test tube with rack, 4. Disposable syringe,
5. Spirit and cotton, 6. Blood sample.
⇒ Description of the apparatus-

Westergren ESR tube:
◊ Length - 300 mm or 30 cm.
◊ Graduation - 0 to 200 in mm.
◊ Open at both ends.
⇒ Procedure-
1. Take 0.4 ml of 3.8% Sodium citrate solution in a test tube.
2. Add exactly 1.6 ml venous blood in the test tube and mix well.
3. Then the citrated blood in sucked into the Westergren ESR tube upto the "0" mark.
4. Place the tube in a stand in a vertical position with the "0" mark above, and allow to
stand for one hour.
5. At the end of one hour the height of the clear plasma is taken from the graduation.
This reading gives the ESR in mm in first hour.
⇒ Result-
ESR of the blood is ....... mm in 1st hour.
**Q- 14. What is thrombocytopenia? What are the causes of thrombocytopenia?

Answer. Thrombocytopenia: It is defined as, “a reduction in the peripheral blood platelet
count below the lower normal limit of 150,000/mm3 of blood (150×109/L).
Causes of thrombocytopenia/Classification:
Acquired-
1. Idiopathic thrombocytopenic purpura (ITP): Primary thrombocytopenia
2. Secondary thrombocytopenia:
a. Drugs and chemicals-
i. Drugs causing aplastic anaemia, e.g. cytotoxic drugs, chloramphenicol.
ii. Drugs causing immune thrombocytopenia, e.g. quinine, chloroquine, quinidine,
sulphonamides, trimethoprim, penicillins, celphalosporins.
b. Infections- Dengue fever, HIV.
c. Aplastic anaemia
d. Acute leukaemia
e. Systemic lupus erythematosus- autoimmune disease
f. Bone marrow infiltration- Secondary carcinoma, malignant lymphomas,
multiple myeloma.
g. Hypersplenism
h. Liver diseases, massive blood transfusion, DIC (Disseminated intravascular
coagulation), post-partum thrombocytopenia.
Neonatal and congenital thrombocytopenia-

1. Immune: Autoimmune and iso-immune.
2. Infections: Congenital or neonatal.
3. Drug administration to mother.
4. Congenital megakaryocytic hypoplasia.
5. Hereditary: Sex-linked or autosomal.
6. Congenital leukaemia.
7. Giant haemangioma.
Q- 15. Short notes on: i. APTT.

*ii. Polycythaemia.
*iii. Pancytopenia.
*** iv. MCV.
v. Myeloblast and lymphoblast
vi. Bone marrow aspiration
vii. Laboratory Diagnosis of ITP
Answer. i. APTT (Activated Partial Thromboplastin Time)/ PTTK (Partial

Thromboplastin Time with Kaolin):
Principle-
The test measures the clotting time of plasma after the activation of contact
factors (Kaolin or other surface acting agents which activate the surface clotting factors XII
and XI) but without added tissue thromboplastin. It indicates the efficiency of “intrinsic
pathway.”
Reagents-
a. Plasma
b. Kaolin
c. Phospholipids
Procedure-
1. Mix 0.5ml of phospholipid reagent with equal volume of Kaolin suspension in a test
tube and keep in a water bath at 37°C.
2. Place 0.1ml of control and test (patient’s) plasma in Tubes marked “C” and “T”
respectively
3. Add 0.2 ml of Kaolin phospholipid solution in the tube marked “C”, mix the
contents and start the stopwatch immediately. Keep in a water bath at 37°C for
exactly 10 minutes with occasional shaking. Then 0.1 ml of pre-warmed CaCl2
solution is added and stopwatch is started. The time taken by the mixture to clot is
recorded.
4. Same procedure is repeated with the tube marked “T” and the time required for
clotting is recorded.
Result-
Normally it is 35-45 seconds. An abnormal result is indicated when clotting
time of patient is 10 or more seconds more than the control time.
Significance of the test-

1. It is prolonged in deficiencies of factor V, VIII, X, XI, XII or II. Factor VII
deficient plasma provides a normal result.
2. When one stage prothrombin test is normal, APTT is prolonged in either factor VIII
or factor IX and rare deficiencies of factors XII and XI, prekallikrein or HMW
Kininogen and also the presence of inhibitors.
3. APTT is prolonged in certain diseases, e.g. DIC, liver diseases, massive transfusion
with store blood, administration of heparin and circulating anticoagulants.
4. Generally APTT is a very sensitive test for the diagnosis of Haemophilia A and B.
*ii. Polycythaemia: Polycythaemia is defined as “an increase in the number of RBC

per unit volume of blood above the normal for age and sex of the patient.”
Causes/ Types of Polycythaemia –
A. True polycythaemia:
1. Primary Polycythaemia/ Idiopathic Polycythaemia Vera
2. Secondary Polycythaemia (Erythrocytosis)-
a. Secondary to hypoxia, e.g. High altitude, Congestive Heart Disease,
COPD.
b. Secondary to erythropoietin production, e.g. hydronehprosis, renal
ischaemia.
3. Benign familial polycythaemia.
4. Polycythaemia associated with haemoglobin disorders.
B. Relative polycythaemia:
1. Dehydration due to severe vomiting, Diarrhoea, burn.
2. Redistribution of body fluids.
3. Pseudopolycythaemia due to stress.
*iii. Pancytopenia: Pancytopenia is the state where all the three formed elements of
blood are reduced, i.e. there is anaemia, leucopenia and thrombocytopenia.
Causes of Pancytopenia-
1. Aplastic anaemia
2. Administration of cytotoxic agents
3. Radiotherapy
4. Overwhelming infections
5. Subleukaemic acute leukaemia
6. Bone marrow infiltration- Lymphomas, metastatic carcinoma of bone, multiple
myeloma, myelofibrosis.
7. Hypersplenism
8. Megaloblastic anaemia
9. Systemic lupus erythematosus
Laboratory diagnosis-
a. Routine Blood Examination
b. Bone marrow examination
c. Other investigations depending on clinical diagnosis.
*** iv. Mean corpuscular volume (MCV): This is the mean

or average volume of a single red cell expressed in femtoliter.
PCV l/l
Calculation- MCV = femtoliter
RBC count per liter
PCV % × 10
= femtoliter
RBC (1012/L)
Normal value- Adult: 76 – 96 fl (1 fl= 10-15 L)

Infant: 106 fl (1 fl= 10-15 L)
Children: 65 – 75 fl (1 fl= 10-15 L)
Significance-
1. It increases in macrocytic anaemia.
2. It decreases in microcytic anaemia.
.
v. Myeloblast and lymphoblast: Difference between myeloblast and lymphoblast-

Parameter Lymphoblast Myeloblast
a. Cell size Smaller Larger
b. Nucleoli number 0-2 2-4
c. Auer Rod Absent Present
d. Myelobperoxidose Negative Positive
stain
e. PAS stain Positive Negative
f. Sudan black B Negative Positive
g. CD markers Positive CD 9,22,10 (B cell) & CD 7,3 (T CD 13,33
cell)
vi. Bone marrow aspiration: Indications of bone marrow aspiration-

1. Leukaemia
2. Anaemia especially Megaloblastic Anaemia and Aplastic anaemia.
3. Subleukaemic acute leukaemia.
4. Aleukaemic leukaemia.
5. Pancytopenia
6. Myelofibrosis and myelodysplastic syndrome
7. Multiple myeloma
8. Secondary carcinoma or metastatic cancers in bone marrow
9. Storage diseases like G6P DHD, Niemannpick disease, Gauchers disease
10. Parasite like LD bodies.
vii. Laboratory diagnosis of ITP (Idiopathic Thrombocytopenic Purpura):

1. Platelet count- Reduce. Below normal to less than 10,000/ cu. mm.
2. Bleeding time- Prolonged to 30 minutes or longer.
3. Coagulation time- Normal
4. Tourniquet test (Capillary resistance test of Hess)- Positive.
5. Blood film- a) Platelet may be abnormal: large, small and atypical forms.
b) Red cells: Normocytic normochromic.
6. Haemoglobin- Reduced.
7. WBC count- Normal.
8. ESR- Normal
9. Bone marrow- Megakaryocytes and their precursors are normal or increased
in number. Otherwise the bone marrow normal.
10. Anti-platelet antibodies may be demonstrated.
“The man who makes his strides in search of knowledge, Allah makes his
path easier towards Heaven.” Al-Hadith.
“He who is worried can not smile, only though wisdom can his sorrow be
wiped out. You will be a man of bad luck, if you are jealous of others’ good luck.
You should establish yourself in virtue, if you want to be successful in your life.
Good luck is always with you, if you pay attention to it. Bodily, vocal and mental
purification is the essence of life. If you want to be happy in this long life, first you
should purify yourself, look within. Nobody can help you except yourself. Poverty
does not matter, matters generosity and hospitality. One who has self purification
is best among men and deity.” Buddha
Blood Transfusion
***Q-1. Define blood group. Name the important blood groups. Why ABO blood group is
called classical blood group?
Answer. Blood group: Blood groups are the groups of blood classified according to the
presence or absence of genetically inherited antigens on the surface of the cells and
antibody in plasma. It can be defined as, “the antigenic make up present on various cellular
and/or soluble components of blood together with the antibody and their interactions.”
Important blood groups:
1. ABO 8. Lewis
2. Rhesus (Rh) Produce 9. Diego Produce
3. MNS HTRs & 10. Dombrock Delayed Haemolytic Transfusion
4. Duffy HDN 11. Indiana Reactions (DHTRs)
5. Kell 12. Colton
6. Kidd 13. Xg
7. Lutheran 14. P
Importance of blood groups:

1. For safe and proper transfusion of blood.
2. To diagnose immune haemolytic anaemia.
3. Investigations of haemolytic transfusion reactions (HTRs).
4. Relationship of blood groups and susceptibility/characteristics/development of
diseases.
5. Medico-legal, Anthropological and Ethnological Research.
ABO systems/ ABO blood groups are called classical blood groups, because-
1. These are the principle blood groups and found among all the people.
2. They maintain both first and second part of Landsteiner’s Law.
3. The hazards of mismatched blood transfusion of ABO blood groups are most
effective and appear more quickly than others.
***Q-2. What are the methods of blood grouping? Describe the slide method of blood
grouping.
Answer. The methods of blood grouping: There are two methods-
1. Slide method
2. Tube method
Blood Grouping by Slide method:

1. Take a clean glass slide and divide into three halves by a marker pen and label
them as A, B and D.
2. Place a small drop of anti-A serum on the area marked A, anti-B serum on the
area marked B and Anti-D serum on the area marked D.
3. Place one drop of blood in each half of the slide from a finger prick and add one
to two drops of normal saline immediately to each drop. Or alternatively, add
one drop of 5-10% red cell suspension in normal saline to each half of the slide.
4. Mix the contents of the each half using separate applicator sticks or glass rods
and rotate the slide gently.
5. After 2-8 minutes the mixture is observed for any agglutination and clumping
both macroscopically and microscopically.
6. The interpretation is done from the following chart, where “+”= agglutination
and “-” = no agglutination.
Area- “A” Area-“B” Area- “D” Blood
(Red cell and anti-A serum) (Red cell and anti-B serum) (Red cell and anti-D Group
RBC+ α agglutinin RBC + β agglutinin serum)
+ - + A+ve
+ - - A-ve
- + + B+ve
- + - B-ve
+ + + AB+ve
+ + - AB-ve
- - + O+ve
- - - O-ve
**Q-3. Give the indication and contraindication of blood transfusion. What do you mean
by Safe Blood Transfusion?
Answer. Indication of blood transfusion:
A. Whole blood-
1. Traumatic, surgical blood loss
2. GIT and uterine haemorrhage
3. Obstetrical haemorrhage
B. Packed cell-
1. Chronic anaemias, e.g. thalassaemia, sickle cell anaemia, aplastic anaemia.
C. Granulocyte concentrate-
1. Severe neutropenia, not responding to antibiotic therapy.
2. Post chemo-irradiation.
D. Platelet concentrate-
1. Severe thrombocytopenia with haemorrhage
2. Patient with myelotoxic drug therapy.
3. Aplastic anaemia.
E. Plasma-
1. Frozen fresh plasma (FFP): burns, coagulation disorders
2. Cryoprecipitate (rich in factor VIII & IX): haemophilia A and B.
F. Other conditions where blood transfusion is given-
1. Exchange transfusion in HDN.
2. Hypovolumic shock
Contraindication of blood transfusion:

1. Congestive cardiac failure (CCF)
2. Shortness of breathing
3. Hyperthermia
4. PRV (Polycythaemia Rubra Vera)
5. Circulatory overload
6. Coronary artery disease
7. Pulmonary oedema
8. Hypertension
9. Bilateral renal failure
10. Polycythaemia.
Safe blood transfusion: Safe blood transfusion is the process by which blood is
collected from low risk donors and transfused after necessary testing and proper
screening in accordance with the standard operating procedure.
In simple words, safe blood transfusion means attainment of the following
objective-
1. Selection of proper donor, i.e. low risk donor.
2. Proper cross matching of the donor’s blood and the recipient’s blood.
3. Proper screening of the blood, i.e. checking blood for transmissible diseases
and quantity and quality of the blood.
4. Rational use of blood.
5. Supply of blood with standard quality and quantity.
Q- 4. Give the pathogenesis, treatment and prevention of Erthroblastosis foetalis.

Answer. Erythroblastosis foetalis: Hemolytic Disease of the Newborn (HDN) also known
as erythroblastosis foetalis, isoimmunization, or blood group incompatibility, occurs when
fetal red blood cells (RBCs), which possess an antigen that the mother lacks, cross the
placenta into the maternal circulation, where they stimulate antibody production and the
antibodies return to the fetal circulation and result in RBC destruction. So, it is the
condition in which the lifespan of foetal RBCs are shortened through haemolysis by
maternal allo-antibody against paternal antigen present in foetal RBCs.
Pathogenesis of Erythroblastosis foetalis:

1. Fetus inherits blood group antigens (usually Rh D antigen or ABO antigens) from the
father that are foreign to the mother.
2. Fetal blood gets into mother’s circulation (either during last trimester of pregnancy,
when cytotrophoblast is no longer present or in abortion, miscarriage, during childbirth).
3. Mother makes antibodies to these blood group antigens.

4. Antibodies cross the placenta, attack the foetal red cells, causing hemolytic anemia and
its consequences.
Treatment of erythroblastosis foetalis:

1. Exchange transfusion to all severely affected infants, i.e. replacement of infants
blood usually with O-negative blood.
2. Mild to moderately affected infants are treated according to clinical conditions.
Prevention of erythroblastosis foetalis:

1. Anti-D immunoglobulin prophylaxis should be given to all rhesus negative women
who have not already been sensitised. It can be given soon after delivery and
antenatally at 28 and 34 weeks. Treatment is also indicated after other sensitizing
events such as abortion, miscarriage, amniocentesis, ectopic pregnancy, abdominal
trauma.
2. Transfusion of accurate blood to mother or prospective mothers.
***Q-5. Give the hazards/complications of blood transfusion.

Answer. Hazards/Complication of blood transfusion:
1. Acute Transfusion Reactions-
a. Non-haemolytic febrile reaction
b. Allergic reaction
c. Anaphylactic reaction
d. Acute haemolytic reaction
e. Transfusion associated lung injury (TRALI)
f. Circulatory overload
g. Reactions due to bacterial contamination
h. Air embolism
i. Bio-chemical upsets, e.g. reduction in plasma potassium.
2. Delayed Transfusion Reactions-
a. Delayed haemolytic reaction
b. Post-transfusion purpura
c. Graft versus host disease (GVHD)
d. Transfusion transmitted infections
e. Iron overload
f. Thrombophlebitis
g. Multiple microembolism
***Q-6. Name the different plasma products. Give the indication, contraindication and risk
of fresh frozen plasma.
Answer. The different plasma products are:
1. Fresh Frozen Plasma
2. Cryoprecipitate
3. Intragam
4. Normal Immunoglobulins
5. Hyper Immunoglobulins
6. Anti-D
7. Albumex 20
8. Albumex 4
9. Biostate (Factor VIII Concentrate)
10. Prothrombinex HT (factor II, IX, X) / Prothrombin complex concentrate (PCC)
Indication of Fresh Frozen Plasma:

1. Replacement of coagulation factor deficiency (factor II, IX, X)
2. Thrombotic thrombocytopenic purpura (TTP)
3. Reversal of warfarin
4. Acute Disseminated intravascular coagulation (DIC)
5. Treatment of immunodeficiencies
6. Antithrombin III deficiency
7. Massive blood transfusion
8. Liver diseases
9. Unknown benefits- a. Newborn sepsis without DIC
b. Intraventricular haemorrhage
Contraindication of Fresh Frozen Plasma:

1. Blood volume expansion
2. Wound healing
3. Nutritional support
4. Patients with non-bleeding conditions
5. Patients with no indication
6. Severe anaemia
7. Thalassaemia
8. Massive blood loss after surgery or trauma.
Risk of Fresh Frozen Plasma:

1. Allergic reaction/ Anaphylactic reaction
2. Transmission of disease
3. Haemolysis
4. Fluid overload
5. Excessive intravascular volume
6. Alloimmunization
7. Transfusion associated lung injury (TRALI)
8. Surgical wound infections
***Q-7. What are the types of platelet concentrate? Give the indication and
contraindication of platelet transfusion.
Answer. The types of platelet concentrate:
1. Platelet concentrate prepared from whole blood donations-
a. Single donor unit: It is in a volume of 50–60 ml of plasma which should contain
at least 55 x 109 platelets, <1.2 x 109 red cells, <0.12 x 109 leucocytes.
b. Pooled unit: platelets prepared from 4 to 6 donor units ‘pooled’ into one pack to
contain an adult dose of at least 240 x 109 platelets.
2. Platelet concentrate preparation collected by plateletpheresis.
Indication of platelet concentrate:

A. Decreased platelet production.
1. Malignancy with cytoreductive drugs
2. Aplasia
B. Platelet sequestration, e.g. in splenomegaly.
C. Platelet destruction, e.g. ITP
D. Platelet qualitative defects, mostly when associated with thrombocytopenia.
E. Treatment of bleeding due to-
1. Thrombocytopenia, e.g. haemorrhagic dengue fever.
2. Platelet defects
F. Prevention of bleeding due to thrombocytopenia, e.g. in bone marrow
failure.
Contraindication of platelet concentrate:

1. Idiopathic autoimmune thrombocytopenic purpura (ITP)
2. Thrombotic thrombocytopenic purpura (TTP)
3. Untreated disseminated intravascular coagulation (DIC)
4. Thrombocytopenia associated with septicaemia, until treatment has
commenced or in cases of hypersplenism.
5. Not generally indicated for prophylaxis of bleeding in surgical patients,
unless known to have significant pre-operative platelet deficiency
6. Pre-eclampsia and platelet mediated microangiopathies.
Q- 8. What is blood bank? What are the importances of a blood bank?

Answer. Blood bank: It is the bank of blood or blood components, gathered from
appropriate and healthy donor, stored and preserved for later use for blood transfusion. The
term "Blood bank" typically refers to a division of a hospital laboratory where the storage
of blood product occurs and where proper testing is performed to reduce the risk of
transfusion related events. This includes compatibility testing for transfusion and may
include blood donation processing, depending on the capabilities of the facility.
Importance of blood bank: The most important role reflecting the importance of
blood bank are-
1. Collection of safe blood from healthy donor.
2. Proper preservation of the blood and blood components for future use.
3. Manufacture of blood components.
4. Supply of appropriate blood to patients to save lives.
5. Ensuring rational use of blood.
6. Motivating and encouraging volunteer donor to donate blood.
7. To prevent the transfusion transmitted diseases by screening the blood.
8. Conduct clinical research on blood and its components.
9. Assisting the clinicians and surgeons in patient care, treatment and surgery.
*Q-9. Name the blood constituents available for clinical use. Give the immune
complication of blood transfusion.
Answer. The blood constituents available for clinical use are:
1. Cellular elements-
a. Red cells
i. Red cell concentrated/ Packed red blood cell concentrate
ii. Red cell suspension with normal saline
b. Leukocytes, e.g. Granulocyte concentrate
c. Platelets, e.g. Platelet concentrate
2. Plasma and plasma products-
o Fresh Frozen Plasma
o Cryoprecipitate
o Intragam
o Normal Immunoglobulins
o Hyper Immunoglobulins
o Anti-D
o Albumex 20
o Albumex 4
o Biostate (Factor VIII Concentrate)
o Prothrombinex HT (factor II, IX, X)/ Prothrombin complex concentrate
(PCC)
The immune complication of blood transfusion: There may be-

1. Acute Transfusion Reactions:
a. Febrile reaction
b. Allergic reaction
c. Anaphylactic reaction
d. Acute haemolytic reaction
e. Transfusion associated lung injury (TRALI)
2. Delayed Transfusion Reactions:
a. Delayed haemolytic reaction
b. Post-transfusion purpura
c. Graft versus host disease (GVHD)
d. Immune modulation
**Q-10. Give the indication and contraindication of human albumin.

Answer. Indication of human albumin:
1. Replacement fluid in therapeutic plasma exchange
2. Shock
3. Treatment of diuretic-resistant oedema in hypoproteinaemic patients: e.g.
nephrotic syndrome or ascites.
4. Burns
5. Acute respiratory distress syndrome (ARDS)
6. Cardiopulmonary bypass
7. Haemolytic disease of the newborn (HDN)
Contraindication of human albumin:

1. Cardiac failure
2. Severe anaemia
3. Patients with previous history of incompatibility reaction to such preparation.

4. Intravenous nutritional solution.
Risks/Adverse reactions:
1. Nausea
2. Chills
3. Fever
4. Headache
5. Hypotension
*Q-11. Give the indication and contraindication of hydroxyethyl starch.

Answer. Indications of hydroxyethyl starch: 1. Replacement of blood volume
2. Shock
Precautions: 1. Coagulation defects may occur

2. May precipitate volume overload and heart failure
Contraindications: 1. Do not use in patients with pre-existing disorders of

haemostasis and coagulation.
2. Do not use in patients with established renal failure.
Side-effects: 1. Minor allergic reactions due to histamine release

2. Transient increase in bleeding time may occur
3. Hypersensitivity reactions may occur including, rarely, severe
anaphylactic reactions.
4. Serum amylase level may rise (not significant)
5. HES is retained in cells of the reticuloendothelial system; the
long-term effects of this are unknown
Q- 12. Name the methods of detecting antibody in the laboratory. Describe anyone method.
Answer. The presence of (irregular) blood group antibodies is usually detected in one or
more of three ways:
1. Cell and serum ABO typing
2. Cross matching
a. Major cross matching
b. Minor cross matching
These are done in following phases-
i. Saline phase
ii. Albumin phase
iii. Coombs phase
3. Antibody screening test

a. Direct Coombs test
b. Indirect Coombs test
Antibody screening test:

a. Direct Coombs test-
i. Wash 2-5% red cell suspension at least 3 times and decant as completely as
possible.
ii. Take 2 drops of washed 2-5% red cells and take one tube for positive control
(containing 2 drops of weak anti-D serum and 2 drops of 2-5% O+ve red cell
suspensions) and one tube for negative control (containing 2 drops of known
non-sensitized 2-5 % red cells).
iii. Put 2 drops of Anti-human globulin (AHG) serum to the three tubes.
iv. Centrifuge the tubes at 1000 rpm for 1 minute and check for agglutination both
macroscopically and microscopically.
v. If no agglutination is seen, then leave the tubes at room temperature for 5-10
minutes and then centrifuge and then read for agglutination.
Result: Agglutination = Positive
No agglutination = Negative
b. Indirect Coombs test-

i. In a tube mix 2 drops of serum with 1 drop of 2-5% O+ve red cells.
ii. Incubate at 37°C for 30-60 minutes.
iii. Centrifuge the mixture at 1000 rpm for 1 minute and observe for agglutination
or haemolysis, if positive presence of complete antibody.
iv. Wash the cells 3 times with normal saline and decant as completely as
possible.
v. Add 2 drops of AHG reagent to the cell bottom.
vi. Centrifuge the mixture at 1000 rpm for 1 minute and observe for any
agglutination.
Result: Agglutination = Positive
No agglutination = Negative
Q- 13. Give the composition, indication and contraindication of SAG mannitol blood.
Answer. SAG-M( Saline, adenine, glucose, mannitol) blood composition:
Sodium chloride 8.77 g
Glucose monohydrate 9.00 g
Adenin 0.169 g
Mannitol 5.25 g
Water for injection 1000 mL
450 ml of blood is collected n the primary bag containing CPD solution, the plasma
is expressed after centrifugation to empty bag and then the 100 ml of the SAGM solution is
expressed in the primary bag containing red cells. The red cells can be stored for 41 days in
SAGM.
Indication of SAG-M blood:
1. Haemolytic disease of the newborn (HDN).
2. Surgery in neonates
3. For top up transfusions
4. Acute blood loss
Contraindications of SAG-M blood:

2. Burn
3. Normovolaemic patients with no indications.
Q- 14. How do you screen a blood donor? What are the tests done for screening of blood
for transfusion?
Answer. Screening of a blood donor: The following criteria should be met-
1. Age- 18 – 57 yrs.
2. Weight- males ≥ 55kgs females ≥ 45 kgs.
3. Frequency of donation- Not more than once in 3 month.
4. Haemoglobin- ≥ 12.5 gm/dl.
5. Blood pressure- Systolic: 100-120 & Diastolic: 50- 100 mm of Hg.
6. Pulse- 60-90 beats/minute.
7. Temperature- 98 – 98.6° F (Afebrile)
8. Vaccination – No history of recent vaccination.
9. Drug therapies- normally the donor under any medication should not
be selected.
10. Medical ailments- Should be disease free.
11. Surgery- History of no major surgery. After a few weeks of
minor surgery, may be considered with careful analysis of past medical
history.
12. Medical history- There should be no complication in previous
donations.
13. Other conditions- e.g. In pregnancy, lactation, menstruation, the females
are deferred from blood donations.
The screening of blood for Transfusion: The following tests are done for screening
of blood for transfusion-
A. Mandatory Tests: These tests are done to detect-
1. Hepatitis B virus
2. Hepatitis C virus
3. HIV (Human immunodeficiency virus)

4. Syphilis
5. Malaria
B. Additional non-mandatory tests: These tests are done to detect infrequently
transmitted diseases which includes-
1. Cytomegalovirus (CMV)
2. Epstein- Barr Virus (EBV)
3. Parvovirus B19
4. Trypansoma cruzi
5. Leishmania
6. Toxoplasma gondii
7. Microfilaria
8. Brucella
9. Human T Cell Lymphotropic Virus (HTLV)
10. West Nile Virus (WNV)
11. Babesia species
12. Transmissible Spongiform Encephalopathies.
Q- 15. Write short notes on: ***i. Auto-transfusion.

***ii. HLA system.
***iii. Cross matching
*iv. Cryoprecipitate.
v. Natural and immune antibodies.
Answer.
***i. Auto-transfusion/Autologous transfusion: The autologous blood transfusion
is the transmission of patient’s own blood. Autologous transfusion is defined as, “the
infusion of patient’s own blood”.
Categories of Autologous transfusion-
1. Predeposit
2. Haemodilution and short term storage
3. Intraoperative blood salvage
4. Post-operative blood salvage
Indication for autologous transfusion-
1. Requirement of rare blood group type.
2. Presence of unexpected antibodies in the recipient.
3. Prevention of allo-immunization.
4. Religious belief
5. Cardiovascular, orthopaedic, gynaecological, plastic reconstruction surgery.
Contraindications-
1. Evidence of infection
2. Schedule surgery to correct aortic stenosis.
3. Unstable angina
4. Uncontrolled seizure disorder
5. Myocardial infarction or cerebrovascular accident within 6 months.
6. Significant cardiac or pulmonary diseases who have not yet been cleared for
surgery.
7. High grade left main coronary artery disease
8. Cyanotic heart disease
9. Uncontrolled hypertension.
Advantages of Autologous transfusion-
1. Elimination of the risk of disease transmission (AIDS, HBV, HCV, syphilis,
malaria).
2. No risk of haemolytic, febrile, and allergic reactions.
3. To provide fully compatible blood in immunized patients.
Hazards/ Disadvantages of Autologous transfusion-
1. Anaemia and hypovolaemia
2. Loss of working time and difficulties faced by traveling several time to the blood
bank.
3. Units lost when surgery is postponed or cancelled.
4. Sepsis from blood contamination.
5. Cancer dissemination.
***ii. HLA system: Human Leukocyte Antigen (HLA) system or Major

Histocompatibility Complex (MHC) is a cluster of genes located on short arm of
chromosome 6 (6p) whose main physiologic function is to bind peptide fragments of
foreign protein for presentation to antigen specific T cells which is present in the plasma
membrane of leukocytes as well as various other cells and chemically glycoprotein in
nature.
Types and distribution:

Antigenic type Gene that encode the antigens Distribution
Class I HLA- A Present on all nucleated
HLA- B cells and the platelets
HLA-C
Class II HLA- DP Present on B cells,
HLA- DQ macrophages, dendritic
HLA- DR cells, endothelial cells and
fibroblasts.
Class III C2 and C4 component of Remains free on plasma and
complement serves as inactive precursor
Properdin molecules in complement
Factor B of complement pathway system.
Functions of HLA system-

1. Regulation of immune response.
2. Cell to cell interaction in immune response.
3. Role in host defense
4. Responsible for graft rejection.
Clinical significance and application-
1. Organ transplantation
2. Bone marrow transplantation
3. Platelet transfusion
4. Paternity testing
5. Disease association, e.g. ankylosing spondylitis (HLA- B7), narcolepsy (DR2),
insulin dependent Diabetes mellitus (DR3/DR4) etc.
Tests of HLA typing-
1. Complement dependent micro-lymphocytotoxicity (CDC)
2. Mixed lymphocyte culture (MLC)
3. Primed lymphocyte test (PLT)
4. Biochemical and molecular genetic assay
***iii. Cross-matching: Cross matching is the test to establish compatibility

of the blood of the donor and the recipient before transfusion by determining
whether or not any agglutination occurs against each other.
Types of cross matching-
1. Major/ Forward Cross Matching: The donor’s red cells are mixed with the
patient’s serum to detect antibody in the serum of the recipient.
2. Minor/ Reverse Cross Matching: The patient’s red cells are mixed with the
donor’s serum to detect antibodies in the serum of the donor.
Both major and minor cross matching should be done in following phases:
a. Saline phase- at 22°C and at 37°C.
b. Albumin phase
c. Coombs phase
Purpose of Cross matching-

1. Prevention of transfusion reactions.
2. Assurance of maximum safe blood to the patient.
*iv. Cryoprecipitate: It is prepared by thawing a unit of fresh

frozen plasma at 4°C and then recovering the cold precipitated factor VIII protein by
centrifugation. It contains about half of the Factor VIII and fibrinogen in the donated whole
blood: e.g. Factor VIII: 80–100 IU/pack; fibrinogen: 150–300 mg/pack.
Storage-
At –25°C or colder for up to 1 year
Indications –
1. As an alternative to Factor VIII concentrate in the treatment of inherited
deficiencies of:
— von Willebrand Factor (von Willebrand’s disease)
— Factor VIII (haemophilia A)
— Factor XIII
2. As a source of fibrinogen in acquired coagulopathies: e.g. disseminated
intravascular coagulation (DIC).
Administration –
1. If possible, use ABO-compatible product
2. No compatibility testing required
3. After thawing, infuse as soon as possible through a standard blood
administration set
4. Must be infused within 6 hours of thawing
v. Natural and immune antibodies: Blood group antibodies are of two kinds-
1. Natural antibodies: Naturally occurring antibodies are those found in the blood of
people who have not been exposed to foreign blood cell antigens. Naturally
occurring antibodies are usually IgM i.e. complete antibody.
2. Immune antibodies: Immune antibodies arises as a result of an immune response to
blood cell antigens not normally possessed by an individual but acquired by blood
transfusion or transplantation or by transplacental passage of foetal red cells during
pregnancy. Immune antibodies are mostly IgG, i.e. incomplete antibody.
System Antigens Antibodies Type of antibody

ABO A, B, H Anti-A, Anti-B Natural
Anti-H Natural (found in Bombay Blood
Group)
Rhesus C, D, E, c, e Anti-C, Anti- D, Anti-E, Immune
Anti-c, Anti-e.
MNS M, N, S, s Anti-M, Anti-N, Anti-S Natural
P P, P1 Anti-P1 Natural
Lutheran Lua, Lub Anti- Lua Natural and also immune
Lewis Lea, Leb Anti-Lea Natural
System Antigens Antibodies Type of antibody
Kell K, k Anti-K Immune
Duffy Fya, Fyb Anti-Fya Immune
I I, i Anti-I Natural
vi. Effects of mismatched blood transfusion:

1. Renal shutdown
2. Haemoglobinuria
3. Haemolytic reaction
4. Jaundice
5. Chest pain, lumber pain, nausea, vomiting, breathlessness.
o An envious person does not look at those more capable than him and aspire to better
himself; he plots to drag them down to his own level. Jealousy is simply an unwitting
admittance of one's own flaws and failings.
o People who can admire true greatness are happy people. Such admiration also serves to
elevate one's own existence. By opening wide the doors of our hearts we can broaden our
own horizons and strengthen our belief in life. A breakdown in this capacity to praise and
respect others can lead to inflexibility and self-righteousness.
o There may be times when others seem enviable. But others are others and you are you.
Rather than comparing your joys and sorrows to those of others, you should aim to surpass
your limits in the situation you are in right now. Those who can do this are the true victors
in life.
Histopathology and Cytopathology
*Q- 1. Define pneumonia. Give the classification and organisms of pneumonia. Give the
stages of lobar pneumonia/ Give the morphology of various stages of lobar pneumonia.
Answer. Pneumonia: Pneumonia is defined as any inflammation of lung parenchyma. It can
also be defined as, “inflammation of lung parenchyma with or without consolidation”.
Classification of pneumonia-
A. On anatomical site:
a. Lobar pneumonia
b. Bronchopneumonia
c. Interstitial pneumonia
B. Aetiological classification:
a. Bacterial
i. Streptococcus pneumoniae/ Diplococcus pneumoniae
ii. Haemophilus influenae
iii. Staphylococcus aureus
iv. Enterobacteriacae, e.g. Klebsiella pneumoniae.
v. Legionella pneumophila
b. Fungal
i. Histoplasma capsulatum
ii. Blastomyces dermatitidis
iii. Pneumocystis carinii
c. Viral
i. Influenza virus
ii. Measles
iii. Respiratory sincitial virus
iv. Chicken pox
C. On exudation
a. Suppurative pneumonia
b. Fibrous pneumonia
Morphology/Pathology of lobar pneumonia: Lobar pneumonia passes through four stages-

A. Stage of congestion: 1-2 days
a. Macroscopic- The involved lobe is heavy, boggy red and subcrepitant.
b. Microscopic- i. Vascular engorgement.
ii. Intra-alveolar fluid with few neutrophils and numerous
bacteria.
B. Stage of red hepatization: (2nd - 4th days)

a. Macroscopic- The lobe appears distinctly red, firm, airless with liver like
consistency.
b. Microscopic- i. Solid packing of the alveolar space with neutrophils
extravasated red cells and precipitated fibrin.
C. Stage of gray hepatization: (4th – 8th days)
a. Macroscopic- Solid, grayish brown with dry surface.
b. Microscopic- i. Progressive disintegration of leukocytes and red cells
ii. The alveolar exudate is increased in amount with dense
fibrin strands and numerous polymorphs.
D. Stage of resolution: (8 – 10th day)
th
a. Macroscopic- The affected lobe returns to normal size or almost the normal
size.
b. Microscopic- The consolidated exudate within the alveolar spaces
undergoes progressive enzymatic digestion to produce a granular semi-solid
debris that is either reabsorbed, ingested by macrophages or coughed out.
Fate: 1. Usually complete resolution takes place.
2. May develop complications-
i. Suppurative and lung abscess (by type III pneumococcus, klebsiella,
staphylococcus)
ii. Empyema
iii. Organization and fibrosis
iv. Bacteraemia and septicaemia.
***Q-2. Define peptic ulcer (disease). Give the site and complication of peptic ulcer
(disease).
Answer. Peptic ulcer: Peptic ulcers are chronic most often solitary lesion that may occur in
any part of the gastrointestinal tract which is exposed to aggressive action of acid peptic
juices. So, peptic ulcers are chronic ulcerative lesions of the gastrointestinal tract at the
sites exposed to the aggressive action of acid- peptic juices.
Sites of peptic ulcers: The two major ones are duodenal ulcer and gastric ulcer.
1. Duodenum, first part- About 70%.
2. Stomach, usually antrum- About 28%.
3. Barrett’s oesophagus.
4. Meckel’s diverticulum and adjacent ileum with ectopic gastric mucosa.
5. Duodenum, stomach and or jejunum in Zollinger- Ellison syndrome.
6. Gastroenterostomy stoma.
Complication of peptic ulcer:
1. Bleeding
2. Perforation or penetration into an adjacent viscus.
3. Obstruction from oedema or from scarring of the pylorus.
4. Malignant transformation is extremely rare with gastric ulcers and possibly it is an
ulcerative gastric carcinoma form the outset. Malignant transformation does not
occur with duodenale ulcer.
5. Intractable pain.
6. Acquired pyloric stenosis.
***Q-3. How do you diagnose tuberculosis in the laboratory? (Or, Give the laboratory
diagnosis of Tuberculosis).
Answer. Diagnosis of tuberculosis in the laboratory:
1. Mycobacterial culture- which is the gold standard test should be advised for exact
identification of organism and sensitivity of drugs.
2. PCR for Myco. tuberculosis bacilli- more sensitive than AFB because bacterial load
upto 10 can give positive result.
3. Sputum for AFB- only positive when bacterial load is >5000.
4. FNAC/Cytology/Biopsy of the affected organ.
5. Supportive radiology and imaging technique.
6. ELSIA (enzyme linked immunosorbent assay) - although not very specific.
7. Blood routine examination
a. Hb: Anaemia
b. TC: Relative lymphocytosis, monocytosis
c. ESR: Markedly high, mostly above 100 mm in 1st hour.
8. Others:
a. Mantoux test
b. ICT for TB
***Q-4. Define IHD. What are the risk factor and complication of MI?
Answer. Ischaemic heart disease (IHD): Ischaemic heart disease results from myocardial
ischaemia due to an imbalance between the supply (perfusion) and demand of the heart for
oxygenated blood.
(Ischaemia comprises not only insufficiency of oxygenated blood but also reduced
availability of nutrient substrates and inadequate removal of metabolites. )
IHD is classified into four syndromes:
1. Angina pectoris
2. Myocardial infarction (MI) or Heart attack
3. Sudden cardiac death (SCD)
4. Chronic ischaemic heart disease
Risk factors of Myocardial infarction (MI):

A. Major risk factors
a. Non-modifiable
1. Age: more than 55
2. Sex: male are more prone.
3. Family history
b. Potentially controllable
1. Hyperlipidaemia
2. Hypertension
3. Cigarette smoking
4. Diabetes mellitus
B. Minor risk factors

a. Non-modifiable
1. Physical inactivity
2. Obesity
3. Stressful life
4. Post menopausal oestrogen deficiency
5. High carbohydrate diet
6. Excessive coffee drinking
7. Oral contraceptives
b. Potentially controllable
1. Alcohol
2. Lipoprotein Lp(a)
3. Hardened (trans) unsaturated fat intake
4. Chlamydia pneumoniae
Complication of Myocardial infarction (MI):

A. Minutes – hours
1. Arrhythmia (75-95%)
a. Ventricular fibrillation, acute heart failure.
b. Heart block
2. Cardiogenic shock (10%)- associated with large infarct.
3. Thromboembolic complication (15-40%)
a. Mural thrombosis
b. Atrial thrombosis
c. Leg vein thrombosis
B. 3-14 days
1. Rupture of heart
a. Infarction of whole wall thickness.
b. Unusual increased polymorph activity before organization is established.
c. Mural myocardium rupture
d. Papillary muscle rupture causing mitral regurgitation.
e. Intraventricular septum rupture
2. Acute pericarditis
C. Weeks
1. Chronic heart failure.
D. Months
1. Cardiac aneurysm
2. Post cardial infarction syndrome
E. At any time
1. Recurrence of infarction.
***Q-5. Describe the aetiology and microscopic features of acute appendicitis.

Answer. Aetiology of appendicitis: Aetiology may be divided into existing and
predisposing factors.
A. Existing factors-
1. Age: Mostly children less than 10 years and adolescent.
2. Sexes: Both males and females are equally affected.
3. Diet: High protein and fatty diet with a lack in cellulose.
4. Heredity: May have a family history.
B. Predisposing factors-
a. Obstruction:
1. In the lumen-
i. Faecoliths (most common)
ii. Threadworms
iii. Whipworms
2. In the wall-
i. Swelling of submucous lymphoid tissue.
ii. Extension of carcinoma caecum.

3. Outside the wall-
i. Compression by bands
ii. Kinking of appendix
b. Infection:
1. Escherichia coli and Streptococcus faecalis. In tropics sometime Entamoeba histolytica.
Pathology/Morphology of (acute) appendicitis:
A. Macroscopic-
1. Swollen and distended appendix.
2. Serosal covering reddened.
3. Cut section shows- Lumen filled with pus.
4. Mucosa shows area of ulceration.
B. Microscopic-
1. Infiltration of acute inflammatory cells throughout the wall principally in the
submucosa.
2. Erosion of the mucosal surface.
3. Muscle fibres are separated by oedema fluid.
4. Pinkish exudate is present in the lumen.
***Q-6. Define nephrotic syndrome. Give the difference between AGN & nephrotic
syndrome.
Answer. Nephrotic syndrome: It is characterized by massive proteinuria (>3.5gm/day),
hypoalbuminimia, lipiduria, severe oedema (anasarca), hyperlipidaemia.
The difference between acute glomerulonephritis (AGN) and nephrotic syndrome/
Difference between Acute Nephritic Syndrome and Nephrotic Syndrome:
Traits Acute glomerulonephritis/ Acute Nephritic Nephrotic syndrome
Syndrome
A. Common 1. Post streptococcal immunologically 1. Membranous glomerulopathy.
causes mediated disease. 2. Minimal change disease.
2. SLE (Systemic lupus erythematosus). 3. Focal segmental
3. Post infective endocarditis by glomerulosclerosis.
Staphylococcus. 4. Membrano-proliferative
4. Post viral infections- measles, mumps, glomerulonephritis.
chicken pox, Hepatitis B
B. Clinical 1. Periorbital oedema 1. Anasarca (severe oedema).
features 2. Mild to moderate hypertension. 2. Hypertension absent.
C. Laboratory findings
a. Urine volume a. Reduced a. Normal
b. Haematuria b. Grossly visible b. Absent
c. Proteinuria c. Mild to moderate c. Massive (>3.5gm/day)
d. RBC and d. Present d. Absent.
RBC cast in
urine
e. Lipiduria e. Absent. e. Present.
Traits Acute glomerulonephritis/ Acute Nephritic Nephrotic syndrome
Syndrome
f. Plasma f. Normal f. Reduced.
albumin
g. Serum g. Normal g. Raised
cholesterol
h. Blood urea h. May be increased. h. Normal
and creatinine
***Q-7. Name the intestinal ulcer. Give the difference between typhoid & tubercular ulcer.
Answer. Ulcers of the GIT:
A. Ulcers of oesophagus and stomach-
a. Benign:
1. Chronic gastric ulcer (peptic ulcer)
2. Acute gastric ulcer
i. Stress ulcer
ii. Curling ulcer (ulcer due to extensive burn).
iii. Cushing ulcer (ulcers due to trauma or surgery to brain)
b. Malignant:
1. Ulcerative or excavated gastric carcinoma
B. Ulcers of the intestine-
a. Ulcers of small intestine:
1. Duodenal ulcer
2. Typhoid ulcer
3. Tuberculous ulcer
4. Bacillary dysentery ulcers
5. Crohn’s disease
6. Malignant ulcer
7. Peptic ulcer in Meckel’s diverticulum
b. Ulcers of large intestine:

1. Amoebic dysentery ulcers
2. Bacillary dysentery ulcers
3. Malignant ulcers
5. Tuberculous ulcer in the caecum.
Difference between Typhoid ulcer and Tuberculous ulcer/ Tubercular ulcer:

Points Typhoid ulcer Tuberculous ulcer/ Tubercular ulcer
1. Site 1. In the lower part of the ileum, 1. Ileum, caecum and large intestine
affecting Peyer’s patches and upto the rectum, spread along the
solitary lymphoid follicles. lymphatics forming “gridle ulcers”.
2. Long axis 2. Longitudinal 2. Transverse (usually)
3. Shape 3. Round or oval 3. Irregular or oval.
4. Floor 4. Pitted. Formed by muscular or 4. Mamilated. Formed usually by
serous coat. muscle coat.
Points Typhoid ulcer Tuberculous ulcer/ Tubercular ulcer
5. Edge 5. Raised 5. Undermined.
6. Potential 6. Slight congestion 6. Tubercles are seen.
surface
7. Perforation 7. Common 7. Uncommon.
8. Adhesion 8. Absent 8. Present.
between loops
9. Healing 9. Resolution. 9. Fibrosis.
10. Stricture 10. Does not occur. 10. May occur.
11. Microscopic 11. Macrophages plenty. 11. Structure of tubercle.
Hyperplasia of lymphoid and MPS,
and necrosis.
**Q-8. What is cirrhosis of liver? Classify cirrhosis of liver. Give the pathogenesis and
effects of cirrhosis of liver.
Answer. Cirrhosis of liver: 1. It is chronic disease of the liver characterized by diffuse
fibrosis and conversion of normal hepatic architecture into structurally abnormal nodules.
2. Liver (hepatic) cirrhosis is the end-stage condition of liver disease
which results from persistent and progressive necrosis of hepatocytes, followed by fibrosis
and abnormal nodule formation.
Classification of cirrhosis of liver:

A. Based on morphology-
1. Micronodular or regular cirrhosis- in which the preponderance of parenchymal
nodules are less than 3mm in diameter.
2. Macronodular or irregular cirrhosis – in which most of the nodules exceeds 3mm in
diameter.
3. Mixed- Both small and large nodules are present.
B. Based on aetiology-
1. Alcoholic cirrhosis (Nutritional cirrhosis): 30-70%.
2. Post necrotic cirrhosis- (Primary idiopathic & secondary due to bile duct
obstruction): 10%.
3. Pigment cirrhosis (in haematochromatosis).
4. Cirrhosis associated with Wilson’s disease- rare.
5. Cirrhosis associated with α1 anti-trypsin deficiency (pizz cirrhosis)- rare.

6. Cryptogenic cirrhosis: 15-60%.
7. Cardiac cirrhosis: very infrequent.
8. Carcinomatous cirrhosis.
9. Indian childhood cirrhosis.
10. Viral cirrhosis.
11. Cirrhosis associated with galactosaemia: very infrequent.
12. Cirrhosis associated with syphilis (congenital and tertiary): very rare.
C. Based on birth-
a. Acquired (after birth): e.g.
1. Alcoholic cirrhosis
2. Post-hepatic or post-viral hepatitis
3. Cryptogenic cirrhosis (of unknown aetiology).
b. Congenital (in born errors of metabolism): e.g.
1. Pigmented cirrhosis (in haematochromatosis).
2. Cirrhosis associated with thalassaemia.
3. Cirrhosis associated with Wilson’s disease.
Pathogenesis of liver cirrhosis: Chronic inflammation of liver.

↓
Inflammatory cytokines (e.g. TNF, lymphotoxin, FGF-β, IL-1, and platelet derived
growth factor) are produced by stimulated Kuffer cells, epithelial cells, hepatocytes
and bile duct epithelium.
↓
Causes transformation of ITO cells (perisinusoidal hepatic stellar cells) into
myofibroblastic like cells (collagen-secreting cell)
↓
Collagen deposition, type I & III, in the lobule, creating delicate or broad septal
tract.
↓
Loss of fenestration in sinusoidal epithelium and increased vascular resistance in
space of Disse.
↓
Eventual fibrosis.
↓
Remaining hepatocytes stimulated to regeneration.
↓
Proliferate as spherical nodule within the confines of fibrous septa.
↓
So, there is fibrosis, loss of architecture and formation of regenerative nodules.
↓
Cirrhosis of liver
Effects of cirrhosis: 1. Portal vein obstruction leads to-

a. Splenomegaly
b. Ascites
c. Haemorrhages from the sites of portal systemic anastomosis (portosystemic
shunts).
d. Hepatic encephalopathy.
2. Hepatocellular failure.
3. Liver cell carcinoma.
**Q- 9. Define jaundice. Give the laboratory diagnosis of different types of jaundice
(haemolytic & obstructive jaundice).
Answer. Jaundice: Jaundice may be defined as, “yellow discolouration of the skin, sclera
and mucous membrane due to increased bilirubin concentration in the body fluid above
normal.” So, yellow discolouration of the skin, mucous membrane and internal organs is
called jaundice.
Laboratory diagnosis of different type of jaundice:

Points Haemolytic Hepatocellular Obstructive jaundice
Jaundice jaundice
A. Blood Biochemistry
1. Serum Bilirubin Raised, mostly Raised, both Raised, conjugated.
unconjugated. conjugated and
unconjugated.
2. Alkaline Normal Slightly raised High
phosphatase
3. AST (SGOT) Normal High rise Small rise
4. ALT (SGPT) Normal High rise Small rise
5. Prothrombin time Normal Prolonged Prolonged.
6. Serum albumin Normal Low Normal
B. Haematological Findings of Leucopenia in viral
tests haemolytic anaemia. hepatitis
C. Urine
1. Bilirubin Absent Variable Present
2. Urobilinogen Increased Normal or Decreased.
increased.
3. Bile salts Absent Absent Present.
D. Stool
1. Naked eye Dark Normal colour or Pale or normal colour.
pale Bulky, frothy due to
high fat content.
2. Stercobilinogen Increased Decreased Decreased
3. Fat Normal Normal or increased Increased
E. Special tests
1. Viral hepatitis Not found Found in viral Not found
hepatitis
2. Serum α- Not found Found in hepatoma Not found.
fetoprotein
*Q-10. Write down the risk factors, sites and classification of carcinoma of breast.
Answer. Risk factors of carcinoma of breast (aetiology and pathogenesis):
I. Genetic predisposition-
BRCA1 and BRCA2 tumour- suppressor genes are associated with the
occurrence of breast carcinoma. p53 tumour suppressor gene is also
implicated.
II. Hormonal exposure-
1. Length of reproductive life: Risk increases with early menarche and late
menopause.
2. Parity: Breast cancer incidence is more in nulliparous than in multiparous.
3. Age at first child: Risk is increased in women older 30 years at the time of their first
child.
4. Obesity: There is a increased risk in postmenopausal obese women probably due to
synthesis of oestrogen in fat depots.
5. Oestrogen producing ovarian tumours are associated with breast cancer.
III. Other factors-
1. Age: More common over 50 years and rare before 25 years.
2. Proliferative breast disease: It is associated with an increased risk.
3. Diet: Dietary fat is implicated.
4. Alcohol: Moderate or heavy alcohol consumption is associated an increased risk.
5. Radiation: Exposure to radiation increases the risk.
6. Race: More common in America and in Europe, mostly whites.
7. Breast feeding: Risk is higher in women who do not breast fed their children.
Sites of Carcinoma of Breast:

1. Upper and outer quadrant - 60%
2. Upper and inner quadrant - 12%
3. Central - 12%
4. Lower and inner quadrant - 06%
5. Lower and outer quadrant - 10%.
Classification of carcinoma of breast:

A. Carcinoma in Situ (Non-invasive)-
1. Ductal carcinoma in situ (DCIS)
2. Lobular carcinoma in situ (LCIS)
B. Invasive (infiltrating) Carcinoma-

1. Invasive ductal carcinoma, not otherwise specified (NOS), Scirrhous
carcinoma.
2. Medullary carcinoma
3. Invasive lobular carcinoma
4. Tubular cribriform carcinoma.
5. Colloid or mucinous carcinoma.
6. Papillary carcinoma
7. Adenoid cystic carcinoma.
8. Secretary carcinoma
9. Inflammatory carcinoma
10. Metaplastic carcinoma
*Q-11. Give the aetiology, stages & pathogenesis of carcinoma of cervix.

Answer. Aetiology of carcinoma of cervix:
A. Risk factors-
1. Early age at first intercourse.
2. Multiple sexual partners.
3. A male partner with multiple sexual partners.
4. Increased parity
5. Race: More in white women than black. Less in muslims, jews and nuns.
6. Other factors: Low socio-economic class, family history, immune status,
cigarette smoking, contraceptive use, prostitution and multiple marriages.
B. Suspected causative agents-

1. Human papilloma virus (HPV): HPV is now considered an important factor
in cervical oncogenesis. Low risk HPV types 6 and 11 are associated with
condyloma acuminatum (benign tumour) and high risk HPV types 16,18, 31
and 33 are associated with cervical intraepithelial neoplasia (CIN) and
invasive carcinoma.
2. Herpes simplex virus type II (HSV II).
Stages of carcinoma of cervix: The following stages of carcinoma of cervix have

been clinically recognized-
Stage O : Carcinoma in situ.
Stage I : Carcinoma confined to the cervix.
Stage II : Carcinoma extending beyond the cervix but not reaching the pelvic wall.
State III : Reaching upto the pelvic wall.
Stage IV : Reaching the bladder and the rectum with widespread metastasis.
Pathogenesis of carcinoma of cervix: Sexual activity

↓
HPV exposure
↓
Cervical transformation zone
Squamous differentiation Endocervical columnar

differentiation
Squamous intraepithelial
lesion
Low grade: High grade:

Low risk HPVs High risk HPVs Glandular
intraepithelial lesion:
(adenocarcinoma in situ)
High risk HPVs
(16,18, 31, 33, 45)
Rare Smoking, oral contraceptives, high parity,

altered gene and immune status
Invasive Squamous
Carcinoma Invasive
Adenocarcinoma
Figure- Pathogenesis of cervical carcinoma
***Q-12. Give the advantages, disadvantages and indication of FNAC.

Answer. Advantage of FNAC (Fine needle aspiration cytology)/ FNAB (Fine needle
aspiration biopsy)/ ABC (Aspiration biopsy cytology):
1. It is quick, convenient, and economic procedure which can be practised on a
outpatient basis.
2. Local anaesthesia is not required and less traumatic to patients.
3. It is rapid and results can be obtained quickly.
4. It can be attempted at multiple sites and can be repeated if necessary.
5. Investigation of a mass is possible without excision.
6. Provides preoperative knowledge of deep steated lesions.
7. It can be done in patients where surgery is contraindicated.
8. Aspirated material can be used for bacteriological examination and
immunochemistry, flow cytometery, PCR and other techniques.
9. The procedure can be done for follow up cases and further management.
Disadvantages of FNAC:
1. Danger of dissemination of tumour cells in the needle tract or natural cavities.
2. It may cause internal bleeding and complication like air embolism,
penumothorax.
3. Accuracy of the method has been questioned- the point of the needle may miss
the target.
4. The procedure has the following limitations-
a. Considerable training and expertise is needed to rely on the diagnosis.
b. The procedure is not suitable for many non-neoplastic conditions where
preserved detail tissue architecture is necessary for diagnosis.
c. False negative results may be obtained in the following situations:
i. If there is extensive fibrosis and sclerosis in a tumour.
ii. If the tumour is highly vascular.
iii. If there is tumour necrosis.
Indication of FNAC:
1. When surgery in not possible-
a. Patient refuses surgery or tolerates anaesthesia poorly.
b. Exploratory mass in a poor risk patient.
c. Difficult or dangerous site of location of mass.
2. When surgery is contraindicated-

a. Clinically an obvious malignant mass.
b. Clinically the mass is a cyst or obviously benign.
c. Age of patient and other factors, e.g. pregnancy.
Application of FNAC: It is applied for diagnosis of palpable as well as non-

palpable lesions.
I. Palpable mass lesions in:
1. Lymph nodes
2. Breast
3. Thyroid glands
4. Salivary glands
5. Soft tissue masses
6. Bones
II. Non-palpable mass lesions in:
1. Abdominal cavity
2. Thoracic cavity
3. Retroperitoneum
***Q-13. How will you prepare a slide for histological examination?

Answer. The following steps are done for processing of tissue for histological examination-
1. Grossing and labelling of the specimen
2. Fixation: Fixation is done overnight at room temperature in 10% Formalin or in
Carnoy’s fixative for 1-3 hours at room temperature.
3. Dehydration: The material is kept in ascending concentration of alcohol, e.g. 50%,
60%, 70%-100% for 1 hour each. It is not done suddenly because it may hamper
cell structure.
4. Clearing: The tissue is kept in Xylol for 1 hour to remove the alcohol and increase
the translucency.
5. Paraffin impregnation and embedding: The tissue is kept in paraffin bath containing
melted paraffin at 50°-70° C for 1 hour. After this the melted paraffin is poured in a
small base and the tissue is put in it in proper plane. Allow to cool and harden the
paraffin in room temperature. Take out the paraffin block and keep the block in the
ice chamber of the refrigerator for some time before section cutting.
6. Section cutting: In this stage, the tissue is sectioned form 3-5 micron in thickness by
a microtome machine. A water bath with a temperature 45°-50°C is used for this
floatation of sections. Sections are cut and selected sections are placed on the water
bath and then taken on the glass slide(s) previously smeared with egg albumin. The
slides are kept in slanting position for some time to drain out the water and allowed
to dry at room temperature.
7. The dried up slide containing the sectioned tissue is placed in a paraffin oven at a
temperature of 60°-65°C for 15 minutes. Then the slide is put in Xylol I for 5
minutes and in Xylol II for next 5 minutes to remove the paraffin.
8. The slide is treated with descending grade of alcohol, i.e. 100%, 90%, 80% - 50%
for 5 minutes each to remove Xylol. Lastly wash with tap water for 2 minutes.
9. Staining: Two stains are used for this purpose. Haematoxylin (basic dye) and Eosin
(acid dye). First the slide is kept in the haematoxylin for 10-15 minutes and this
stains the nucleus blue. The slide is washed under running tap water for 5-10
minutes. Then the slide is given 2-3 quick dips in 1% acid alcohol to differentiate
which nuclei do not stain with haematoxylin and then washed with water. After this
the slide is counter stained by eosin for 20 seconds to 2 minutes depending on the
age of the eosin and the depth of counter stain required. Wash under running tap
water till eosin is differentiated, eosin stains the cytoplasm red colour.
10. Dehydration: The slide is again treated with ascending grade of alcohol for 2
minutes for removal of the remaining water.
11. Clearing: The slide is placed in xylene for 5-15 minutes. The slide is cleared and
dried up.
12. Mounting: One drop of DPX or Canada balsam is placed on a coverslip and placed
it on the sectioned tissue and allowed it to dry.
13. Place the slide for microscopic examination.
Result-
Cell nucleus - Blue
Cytoplasm - Pink
Collagen fibers - Pink
Reticulin fibers - Pink
RBC - Bright red
WBC - Nucleus blue, cytoplasm vary with cell type.
Q- 14. Define AGN. Write down the pathogenesis of AGN.

Answer: Acute glomerulonephritic syndrome (AGN)/ Nephritic syndrome/ Type I
Nephritis/ Proliferative Nephritis: It is characterized by inflammation in the glomeruli
which is manifested by acute onset, grossly haematuria, mild to moderate proteinuria,
hypertension and usually follow acute post streptococcal glomerulonephritis.
Pathogenesis of AGN:
It is mediated by type III hypersensitivity reaction. In response to the above antigen
(streptococcal or non-streptococcal) antibody is formed→ deposition of circulating
immune complex (ag+ab) in glomeruli→ ag+ab+c (complement mediated injury) →
Neutrophils come to the area→ Phagocytosis of immune complex. Some cell die and
release of lysosomal enzymes→ damaging the endothelial cells and glomerular basement
membrane (GBM)→ In response to injury endothelial cells and mesaginal cells proliferate
(cellular swelling) later→ epithelial cell proliferation→ Acute glomerulonephritis.
*Q- 15. What are the sites of tuberculosis? Give the difference between primary &
secondary tuberculosis/Give the difference between primary & post primary tuberculosis.
Answer. The sites of Tuberculosis:
1. Lungs
2. Kidneys
3. Bones
4. Lymphnodes
5. Brain
6. Intestine
7. Liver and spleen
Difference between primary & secondary tuberculosis/The difference between

primary & post primary tuberculosis:
Points Primary tuberculosis Secondary/Post-primary/ Reinfection
tuberculosis
1. Age 1. Children. 1. Adult
2. Site 2. It usually affect in the lower part of 2. It begins in the apical or post
the upper lobe or upper part of lower segments of one or both upper lobes.
lobes of one lung- Bilateral or
multiple foci are very infrequent.
3. Source of 3. Exogenous. 3. Endogenous or exogenous.
infection
4. Lesion:
a. a. The characteristic lesion is the a. The characteristic lesion is
Characteristics presence of Ghon focus. The presence formation of tubercle (focal area are
of Ghon complex (Ghon focus+ of caseous consolidation) without
regional lymph involvement). regional lymph node involvement.
b. Type b. Exudative b. Productive.
5. Fate
a. Fibrosis a. A little or no fibrosis. a. Fibrosis is a conspicuous feature.
b. Spread b. Progressive spread. b. Progressive pulmonary
tuberculosis.
c. Mode of c. Rapid- by a process of exudative c. Slow- by a process of proliferation
spread lesion, along the lymphatics. often along the tissue spaces.
Q- 16. Give the laboratory diagnosis of carcinoma of cervix and breast.

Answer. Laboratory diagnosis of carcinoma of cervix:
1. Papanicolaou cytological test/ Pap smear study: Abnormal cells show cellular and
nuclear pleomorphism.
2. Cervical biopsy and histopathology
3. Colposcopy
4. Schiller’s test- uses iodine solution to stain.
Laboratory Diagnosis of Carcinoma of Breast:
1. FNAC
2. Histopathology- Biopsy, Lumpectomy, mastectomy, axillary tail and lymphnode.
3. IHC for ER & PR receptor, Neu/erb, BRCA1, BRCA2.
4. Mammography
5. Chromosome analysis
6. PCR
7. Radiology and imaging like CT scan, MRI.
Q- 17. Enumerate the causes of rheumatoid arthritis. State the pathology of rheumatoid
arthritis.
Answer. Rheumatoid arthritis: It is a chronic systemic inflammatory disorder that may
affect many tissues and organ- skin, blood vessels, heart, lungs and muscles, but principally
attack the joints, producing a non-suppurative proliferative and inflammatory synovitis that
often progress to destruction of the articular cartilage and ankylosis of the joints.
Causes of Rheumatoid arthritis: The exact causes of rheumatoid arthritis are

unknown. Rheumatoid arthritis is most likely triggered by a combination of factors,
including an abnormal autoimmune response, genetic susceptibility, and some
environmental or biologic trigger, such as a viral infection or hormonal changes.
The key pieces of evidence relating to pathogenesis are-

1. A genetic link with HLA-DR4 and related allotypes of MHC Class II and the T cell-
associated protein PTPN22.
2. A link with cigarette smoking that appears to be causal.
3. A remarkable deceleration of disease progression in many cases by blockade of the
cytokine TNF (alpha).
4. A similar dramatic response in many cases to depletion of B lymphocytes, but no
comparable response to depletion of T lymphocytes.
5. A more or less random pattern of whether and when predisposed individuals are
affected.
6. The presence of autoantibodies to IgGFc, known as rheumatoid factors (RF), and
antibodies to citrullinated peptides (ACPA).
Pathology of Rheumatoid arthritis: Rheumatoid arthritis (RA) is a chronic, systemic

inflammatory disorder that may affect many tissues and organs, but principally attacks
synovial joints. The process produces an inflammatory response of the synovium
(synovitis) secondary to hyperplasia of synovial cells, excess synovial fluid, and the
development of pannus in the synovium. The pathology of the disease process often leads
to the destruction of articular cartilage and ankylosis of the joints. Rheumatoid arthritis can
also produce diffuse inflammation in the lungs, pericardium, pleura, and sclera, and also
nodular lesions, most common in subcutaneous tissue under the skin.
Q- 18. Define atherosclerosis. Write down the risk factors and complications of
atherosclerosis.
Answer. Atherosclerosis (AS): Atherosclerosis is defined as progressive inflammatory
disease of arterial wall characterized by formation of intimal lesion called atheromas or
fibrofatty plaques which produces obstruction and ultimately weakens the underlying
media- the vascular lumen.
Risk factors for the development of Atherosclerosis (AS): These include major risk
factors which may be modifiable or non-modifiable and lesser, uncertain or nonquantitated
factors.
A. Major risk factors-
a. Non-modifiable- like increasing age, male gender, family history, genetic
abnormality.
b. Modifiable or potentially controllable- like hyperlipidaemia, hypertension,

cigarette smoking, Diabetes.
B. Lesser, uncertain, non-quantitative factors-
a. Obesity
b. Alcohol
c. Stress
d. Postmenopausal oestrogen deficiency
e. high carbohydrate intake.
Complication of atherosclerosis/ atheromatous plaque:

a. Atherosclerosis of coronary arteries leads to ischaemic heart disease (IHD).
b. Atherosclerosis of internal carotid and middle cerebral arteries leads to
cerebrovascular disease (Stroke).
c. Focal ulceration, rupture and erosion- this leads to exposure of most potent
thrombogenic sub-endothelial connective tissue that induce thrombus formation.
May discharge fragments, debris into circulation leading to atherothrombi or
cholesterol emboli.
d. Haemorrhage into the plaque especially in coronary arteries. A contained hematoma
may expand the plaque or induce rupture.
e. Thrombosis- the most feared and important complication that follows disrupted
lesions. Sometimes may heal and become incorporated.
f. Aneurysmal dilation may occur due to atrophy of underlying media which cause
weakness and rupture.
g. Renal arteries- atheromatous plaque occurs at the origin of renal artery.
Hypertension may develop.
*Q- 19. Give the pathogenesis & pathology of infective hepatitis with laboratory
diagnosis.
Answer. Pathogenesis of infective hepatitis/ Hepatitis A: There are three possibility exist-
1. A direct cystopathic effect.
2. The induction of immune response against viral antigens that damage virally
infected hepatocytes.
3. Alteration of liver cell antigens and the initiation of an autoimmune reaction. Virus
infected cell→ alter the antigenic structure of cell membrane of hepatocytes by the
following way:
a. by introduction of viral antigens.
b. by exposing the sequestrated antigen.
c. By including the synthesis of new antigen.
Pathology:
A. Macroscopic-
Liver cell is slightly enlarged, swollen, tense and bile stained.
B. Microscopic-
1. “Diffuse Ballooning” degeneration of liver cells.
A. 2. Cord arrangement of liver cell lost because:
a. Swelling of the cells.
b. Regeneration of cells.
3. Focal necrosis- especially in the centriobular region liver cells are cytolysed by the
virus and “dropped out”. These areas then infiltrated with inflammatory cells.
4. Kuffer’s cell hyperplasia with lipofuscin pigment and other debris.
5. Regeneration of hepatocytes in the recovery phage.
6. Bile stasis within the canaliculi of the liver.
7. Portal area shows the infiltration of lymphocytes, plasma cells and monocytes.
8. Councilman’s body- coagulative necrosis of the hepatocyte with disappearance of
nucleus and produce a round dense eosinophilic body known as councilman body found
in HAV.
Fate of viral hepatitis:

Fate of HBV infection-
Viral hepatitis
Chronic HBV infection Acute hepatitis

↓ ↓
1. Chronic persistent hepatitis 1. Asymptomatic carrier state
↓
Recovery without major damage 2. Anicteric
↓
2. Chronic active hepatitis 3. Icteric
↓ ↓
3. Cirrhosis 4. Fulminating
↓ ↓
4. Hepatocellular carcinoma 5. Death or recovery.
Hepatitis A- cent percent cure rate. Rarely <1% it may cause fulminant hepatitis.
In case of viral non-A non-B hepatitis
a. Recovery
b. Chronic hepatitis
c. Fulminant hepatitis
d. Carrier state.
Laboratory diagnosis of Hepatitis:

A. PCR
B. ELISA
C. Haematological tests-
1. Leucopenia with relative lymphocytosis.
2. ESR is raised.
D. Liver function tests- Findings of intrahepatic cholestasis.
1. SGPT
2. SGOT
3. Serum Bilirubin
4. Alkaline phosphatase
5. LDH
6. Prothrombin time
7. Serum A:G ratio
E. Viral markers-
1. Hepatitis A: Antibodies to HAV. Presence of anti-HAV IgM in serum indicates an
acute infection. IgG antibodies are common in general population.
2. Hepatitis B:
a. Antigens-
i. HBsAg: It appears before the onset of symptoms and peaks during overt
disease. In 3-6 months it is usually undetectable.
ii. HBeAg: It rises early and declines rapidly. It indicates viral replication. Its
persistence indicates a chronic liver disease or a carrier state.
b. Antibodies-
i. Anti-HBs: It appears late and indicates immunity.
ii. Anit-HBc: IgM anti-HBc suggests an acute infection.
iii. Anti-HBe: It appearance means decreased infectivity.
Q-20. Briefly describe the Haematoxylin and Eosin stain. Shortly describe the progressive
and regressive stain.
Answer. The Haematoxylin and Eosin stain:
Harris’s Haematoxylin-
Haematoxylin - 2.5 gm
Absolute alcohol - 25 ml
Potassium alum - 50 gm
Mercuric oxide - 1.25 gm
Glacial acetic acid - 20 ml
Eosin-
1% Stock alcoholic Eosin
Eosin Y - 1 gm
Dissolved and add Alcohol 95% - 80 ml
Working solution-
Eosin stock solution (1part) + 80 % alcohol (3parts)
Just before use add 0.5 ml of glacial acidic acid to each 100 ml of stain and
stirr.
Procedure of H & E stain-

1. Deparaffinze in xylol hydrate through graded alcohols to water.
2. Remove fixation pigments if necessary.
3. Stain in Harris haematoxylin for 10-15 minutes.
4. Rinse in tap water until sections are “Blue” (5 minutes or less).
5. Differentiate in 1% acid alcohol, three to ten dips. Check the differentiation with a
microscope. Nuclei should be distinct and the back ground very light or colourless.
6. Wash briefly in tap water.
7. Dip in ammonia water or lithium carbonate water until sections are again blue (3-5
dips).
8. Wash in running tap water for 10-20 minutes.
9. Stain in Eosin for 15 seconds to 2 minutes.
10. Dehydrate in 95% and absolute alcohols until excess eosin is removed.
11. Clear in Xylol and mount in DPX.
Progressive and regressive stain: Progressive stain is done by watching the
degree of staining in sections under the microscope at various points during the staining
process and stopping the process when the selective action of the stain has differentiated
the desired parts.
When regressive staining techniques are employed, all the stainable tissue
components are completely saturated with dye. To have any value as a readable slide, some
of this excess stain must be removed, and the process is called differentiation.
Differentiation usually gives sharp staining contrasts because the hydronium and hydroxide
ions in the solvents used to differentiate diffuse more rapidly than does any dye ion, and
this accounts for the more even results obtained. The differentiation step is a relative one
and removes dye from certain tissue components more easily and rapidly then from others.
A properly destained section will have the desired features retaining sufficient stain to
make them clearly visible, and the other tissue components will be completely cleared from
the dye. Some of the ways in which a section may be differentiated include the use of
acidic or basic medium, excess mordant, buffers, or oxidizers.
It is generally known that basic dyes are differentiated by a weakly acid medium,
and acid dyes are differentiated by a weakly basic one. For example, when staining in alum
haematoxylin, one may use a solution of acid alcohol to differentiate the section. If a
section has been overstained in eosin (an acid dye), it can be differentiated in a basic
medium composed of alcohol containing 0.1%-0.5% concentrated ammonium hydroxide.
Q-21. Name five cytopathological specimens that are commonly received in the
laboratorirs. How will you process pleural fluid sample for cytopathological examination.
Answer. Commonly received cytopathological specimens:
1. Cervical smear (paps smear)
2. Body fluids (e.g. pleural fluid, ascitic fluid, CSF, peritoneal fluid).
3. Sputum
4. Aspiration from lymph nodes
5. Aspiration from glands
6. Urine
7. Aspiration from breast lump
8. Stomach aspirates
Process of pleural fluid for cytopathological examination:

1. Centrifugation-
a. Fix the specimen by mixing it with an equal amount of 95% alcohol.
b. Place the fixed specimens in 50 ml centrifuge tubes, and centrifuge at 2000 rpm for
20 minutes.
c. Decant the supernatant and re-suspend the sediment in the remaining drop of fluid.
Pool the sediment, if necessary. The sediment is now ready for preparation of the
smear.
2. Preparation of smear-
a. Transfer the fixed specimen onto a pre-labelled clean slide and prepare a smear by
streaking or spreading or pull-apart.
3. Fixation-
a. Use of 1:1 ether-alcohol is recommended.
4. Staining-
a. Stain by Papanicoloau stain.
Q-22. Define and classify fixative. Which one is ideal fixative for biopsy specimen and
why? What are the equipments required for gross examination of a large soft tissue tumour.
Answer. Fixative: Fixatives are chemical substances which preserve the cellular structure
and tissue arrangements most closely to the original form present in the body. So, the
fixatives are agents that fix the tissues which prevent autolysis and preserve the tissues by
killing and hardening.
Classification of Fixatives:
A. Classification based on mode of action-
a. Primary fixative: Fixative that can be used for the fixation of tissues for the
first time. e.g. 10% Formalin, Zenker’s fluid.
b. Secondary fixatives: The fixatives that are used after the tissue first been
fixed for 3-4 hours in formalin. e.g. Formol sublimate and Helly’s fluid.
B. Classification based on chemical properties-
a. Simple fixative, that consists of one substance, e.g. 10% Formalin.
b. Compound fixative, that consists of two or more substances, e.g. Bouin’s
fluid, Zenker’s fluid, Carnoy’s fluid.
C. Classification based on practical use-
a. Routine fixative, e.g. 10% Formalin.
b. Special fixative, e.g. Zenker’s fluid, Helly’s fluid, Bouin’s fluid.
D. Special classification-
a. Microanatomical fixative, which preserve the anatomy of the tissue.
b. Cytological fixative, which may be cytoplasmic or nuclear and preserve
the respective intracellular constituents.
c. Histochemical fixative, employed for demonstration of histological
constituents and enzymes.
Ideal fixative for Biopsy: Formalin is considered as an ideal fixative as-
1. It is relatively inexpensive, readily available and easy to prepare.
2. It is compatible with most stains and penetrates the tissues well.
3. Normal colour of the tissue is remained.
4. Best fixative for neurological tissues.
Disadvantages of formalin:
a. Causes excessive hardening of tissues.
b. Causes irritation of skin, mucous membrane and conjunctiva.
c. Leads to formation of formalin pigment in tissues having excessive
blood at an acidic PH which can be removed by treatment of
section with picric-alcohol in solution of NaOH.
The equipments required for gross examination of a large soft tissue tumour:
1. Jar with appropriate fixative
2. Measuring tap
3. Cassettes
4. Knife
5. Tray
6. Marker pen and tagging papers
7. Forceps
8. Spatula
Q-23. Name the malignant tumours of uterine cervix. Which test is done for carcinoma
cervix?
Answer. Malignant tumours/ carcinomas of cervix:
1. Ectocervix (uterine cervix)-
i. Squamous neoplasia:
a. Cervical intra-epithelial neoplasia (CIN)
b. Squamous cell carcinoma
2. Endocervix-
a. Adenocarcinoma
b. Adenosquamous carcinoma
c. Undifferentiated carcinoma.
d. Leiomyoma.
Tests done for carcinoma of cervix:

1. Pap smear (Papanicolaou cytologic test)- Abnormal cells show cellular and
nuclear polymorphism.
2. Cervical biopsy and histology
3. Colposcopy
4. Schiller’s test.
Q-24. Define microtomy. What are the types of microtome? How will you care it?
Answer. Microtomy: The process by which the tissues are cut in thin lamellae or thin
sections of uniform thickness for histological or histopathological study.
Types of microtome:
1. Rotary microtome
2. Rocking microtome
3. Sliding microtome
4. Freezing microtome
5. Base sledge microtome
6. Vibrating knife microtome
Care of microtome:
1. Keep the moving parts well lubricated and clean.
2. Put a cover on the microtome when not in use to prevent dust
accumulation.
3. Do not permit rust, dust or paraffin to accumulate between the
bearing surface of the knife holder, brackets, etc.
4. Surface should be cleaned frequently, then wiped with good neutral
oil (e.g. coconut oil); this will prevent rust formation.
5. After cutting section on the microtome, all accumulated paraffin and
tissue should be removed with a soft brush. Metal parts are cleaned
with xylene (don’t use xylene too frequently, it will remove the
painted finish).
6. All moving parts of the microtome must be lubricated, and kept free
from paraffin. Xylene (or, petroleum ether) helps to remove paraffin.
7. The rigidity of the knife holder and the knife are important but never
adjust any screw so tightly that may cause binding. The instrument
should be tight only to the point of smooth, firm operation.
Q-25. Mention the principle of tissue processing. What are the clearing agents? What are
the criteria for choosing an ideal clearing agent?
Answer. The principle of tissue processing:
a. To prevent post mortem changes such as putrefaction and autolysis.
b. Preserve various cell constituents in as life like manner as possible.
c. Protect by hardening the soft tissues, allowing easy handling during
different steps of processing.
d. Convert the normal semi-fluid consistency of cells to an irreversible semi-
solid consistency. and
e. The visual differentiation of structure by using dyes and chemicals.
Clearing agents: Clearing is the process in which alcohol from tissues and cells is
removed and is replaced by a fluid in which wax is soluble and it also makes the
tissue transparent. Commonly used clearing agents are-
1. Xylene
2. Toluene
3. Benzen
4. Chloroform
5. Ceder wood oil
6. Carbon tetrachloride
7. Amyl acetate
8. Methyl benzoate
9. Inhibisol (Methyl chloroform)
Criteria for choosing an ideal clearing agents:

1. It should be miscible in both alcohol and paraffin wax so that it can replace
alcohol and make room for the paraffin during infiltration and impregnation.
2. It should render the tissue transparent.
3. It should be available and economical.
4. It should be less hazardous and easy to use.
Q-26. Give the laboratory investigation of nephritic syndrome. Give the steps of PAP stain.
Answer. Laboratory investigation of nephritic syndrome:
1. Blood Examination-
a. Blood urea: increased
b. Serum Creatinine: may be increased.
2. Urine Examination-
a. Urine volume: Reduced.
b. Haematuria: Grossly visible.
c. Proteinuria: Mild to moderate.
d. RBC and RBC cast: Present
The steps of PAP stain:

A. Stains-
a. Harris’s Haematoxylin:
Haematoxylin - 2.5 gm
Absolute alcohol - 25 ml
Potassium alum - 50 gm
Mercuric oxide - 1.25 gm
For this purpose, acetic acid is not added.
b. Orange G6 (OG6) solution:
Orange G6 - 0.5 gm
Phosphotungstic acid - 0.015 gm
95% Ethyl alcohol - 100 ml
c. Eosin-Azure 50 (EA 50) solution:
Light green (0.1% in 95% ethyl alcohol) - 45 ml
Bismark brown (0.5% in 95% ethyl alcohol) - 10 ml
Eosin yellowish (0.5% in 95% ethyl alcohol) - 45 ml
Phosphotungstic acid - 0.2 gm
Lithium carbonate (saturated aqueous) solution - 1 drop.
Procedure-
1. Fix the smear in equal parts of ether and 95% alcohol for 30 minutes.
2. After fixation, transfer the slide without drying directly from alcohol-ether solution
to 95% alcohol, then through 80% alcohol, 70% alcohol, and water, to distilled
water.
3. Stain in Harris’s haematoxylin (modified) for 2-3 minutes.
4. Rinse gently in tap water to prevent cells from being washed off.
5. Differentiate carefully the nuclear staining in 1% hydrochloric acid in 70% alcohol;
nuclei should be clear and sharp in detail; the cytoplasm should be light blue and
clear.
6. Place gently running tap water for 5 minutes to wash out the acid thoroughly and to
blue nuclei.
7. Rinse in distilled water and transfer through 70% alcohol, 80% alcohol, to 95%
alcohol.
8. Stain in OG6 for 2 minutes.
9. Rinse five times as follows:
a. Twice in 95% alcohol
b. Once in 1% acetic acid in 95% alcohol.
c. Once in 1% phosphotungstic acid in 95% alcohol.
d. Once in 95% alcohol.
10. Stain in EA50 for 2 minutes.
11. Rinse 9 times as follows:
a. 3 changes of 95% alcohol.
b. 2 changes of absolute alcohol.
c. 4 changes of xylene.
12. Mount in DPX.
Q- 27. Write short notes on: i. Fibroadenoma of breast

**ii. Goiter
iii. Primary complex
iv. Biopsy
v. BCG vaccine
vi. Lymphoma
vii. Bronchogenic carcinoma
* viii. Gall stone
ix. Angina pectoris
x. Pleural effusion
xi. Diabetic ketoacidosis
xii. Oedema
xiii. Malignant cell
xiv. Shock
xv. Hyperplasia
xvi. Granuloma
xvii. Frozen section
xviii. Immunohistochemistry
xix. Decalcification
xx. Special stain
xxi. Quality control in histopathology laboratory
Answer.
i. Fibroadenoma of breast: It is the most common benign tumour of
the female breast arising from both fibrous and glandular tissue.
Incidence of fibroadenoma of breast-
d. Age: It can occur at any age of reproductive life of female but
commonly occurs between 15-30 years.
e. Other factor: It is said to be developed as a result of increased sensitivity
of focal area of the breast to oestrogen.
Morphology-
a. Macroscopic/Grossly: Fibroadenoma is small 2-4 cm diameter, solitary,
well delineated, encapsulated spherical or ovoid mass. The cut surface
show homogenous grey white area with myxoid and slit like space
formed by compressed duct.
b. Microscopically: There is marked proliferation of the fibrous tissue. The
arrangement between fibrous overgrowth and ducts produce two types
of patterns which may coexist in a same tumour-
1. Intracanlicular pattern: where overgrowth of fibrous tissue
compresses the ducts into slit like spaces.
2. Pericanalicular pattern: periductal arrangement of proliferating
fibrous tissue around normal patent or dilated ducts.
**ii. Goiter: It is a disease of the thyroid gland characterized by an enlargement of

the gland, visible externally as a swelling on the front of the neck. In simple goiter the
basal metabolic rate is somewhat lowered, and in toxic goiter it is elevated.
Classification of Goiter: a. Simple goiter
b. Toxic goiter
a. Simple goiter: It is characterized by an enlargement of the entire thyroid gland or
one of its two lobes. It is associated with hypothyroidism, a condition caused by
insufficient production of thyroid hormone. Because the body needs iodine to produce
thyroid hormone, inadequate amounts of iodine in the diet may result in simple goiters.
Simple goiters may be classified as either endemic or nontoxic.
1. Endemic goiters are caused by a deficiency of iodine in the diet and usually occur
in populations living in areas with iodine-depleted soil.
2. The cause of most nontoxic goiters is unknown, but researchers suspect that
environmental factors and heredity play a role. In some areas of the world, certain chemical
compounds in food or water may block the body’s production of thyroid hormones and
lead to nontoxic goiter formation. These compounds, known as goitrogens, also include
certain drugs, such as aminoglutethimide and lithium.
b. Toxic goiter: This includes exophthalmic goiter, hyperthyroidism, thyrotoxicosis,
or Graves' disease, for the Irish physician Robert James Graves, is caused by an excess of
thyroxin secretion.
iii. Primary complex: The typical lesions of primary pulmonary

tuberculosis, consisting of a small peripheral focus of infection, with hilar or paratracheal
lymph node involvement is known as primary complex. Lymphatic spread is the
characteristic of the primary complex. This primary complex remains quiescent in majority
of cases (90%) and in minority, it may progress giving rise to progressive primary
complex, or the case may advance to develop exudative lesions like acute caseous
pneumonia, bronchopneumonia, or acute miliary tuberculosis of the lung or generalized
miliary tuberculosis with localization of various organs. Patients who recover from the
primary infection acquire allergy and immunity and therefore, behave differently in
subsequent infections.
iv. Biopsy: It is the small piece of tissue taken out from a living body for
histoplathological or cytopathological study for investigational purpose (diagnostic biopsy)
or for eradication of a lesion (excisional biopsy). Biopsy can be defined as, “obtaining an
adequate piece of representative tissue which should include both apparently normal tissue
as well as grossly abnormal tissue”.
Types of biopsy-
1. Open biopsy: a. Incisional biopsy
b. Excisional biopsy
2. Endoscopic biopsy
3. Fine Needle Aspiration Biopsy (FNAB)
4. Punch biopsy
5. Trephine biopsy
6. Sigmoidoscopic biopsy
7. Wedge biopsy
8. Drill biopsy
9. Crosby capsule biopsy.
v. BCG vaccine: A freeze dried preparation of attenuated strain of

Mycobacterium tuberculosis (Bacillus Calmette- Guerin), proposed for intradermal
injection in adults and children for immunization against tuberculosis. BCG is only given
to individuals only who are tuberculin negative because BCG can only protect those who
are still unaffected.
According to EPI schedule BCG vaccine should be given at birth or within 3
months after birth.
For adult and children single dose of 0.1 ml intradermally just above the insertion
of deltoid muscle or on the outer surface of the thigh in female baby. For infants single
dose of 0.05 ml.
vi. Lymphoma: Lymphomas are cohesive, solid malignant neoplasms that

arise in lymphoid tissue and are derived from lymphoid cells and rarely histocytes. Sites of
origin may be lymphoid tissue of lymph nodes (most common), bone marrow, GI tract,
spleen, liver, skin or other sites.
Classification of lymphomas- Clinically and histologically:
1. Hodgkin lymphoma (HL)- Sternberg giant cells are present
2. Non-Hodgkin lymphoma (NHL)- Sternberg giant cells are absent.
vii. Bronchogenic carcinoma: It is a primary carcinoma of the lung that

arises from the mucous membrane of the bronchi, bronchioles or alveoli.
Aetiology-
It is responsible for 10 percent of all cancer cases. Males are more affected and the
ratio of male is to female 4:1. Pneumoconiosis, silicosis, asbestosis or siderosis have been
found in association with the carcinoma.
Classification-
1. Gross types: a. Hilar
b. Peripheral or nodular type
c. Diffuse type
2. Histological types: a. Squamous cell carcinoma
b. Adenocarcinoma
c. Small cell carcinoma
d. Large cell carcinoma
e. Mixed / Combine types.
* viii. Gall stone: A gallstone is a crystalline concretion formed within

the gallbladder by accretion of bile components. These are associated with – infection,
stasis of bile and an increase in cholesterol content of the blood.
Classification- A. Aetiological classification:
1. Cholesterol stone
2. Pigment stone
B. Based on composition:
1. Pure gall stone
2. Mixed gall stone
3. Combined gall stone
Complications-
a. Stone in the gall bladder:
1. Acute cholecystitis
2. Chronic cholecystitis
3. Mucocele
4. Empyema
5. Carcinoma
b. Stone in the common bile duct:
1. Obstructive jaundice
2. Cholangitis
c. Stone in the ampula of Vater:
1. Acute pancreatitis
d. Stone in the intestine:
1. Acute intestinal obstruction
ix. Angina pectoris: Angina pectoris is a symptom complex of IHD

characterized by paroxysmal and usually recurrent attack of substernal or pericardial chest
discomfort (variously described as constricting, squeezing, choking, or knife like) caused
by transient (15 seconds to 15 minutes) myocardial ischaemia that falls short of inducing
the cellular necrosis that defines infarction.
Types-
1. Stable / typical angina: Main cause- Atherosclerosis. Not in rest but in exertion.
2. Prinzmetal / variant angina: May be at rest, but no atherosclerosis. This is due to
coronary vasospasm.
3. Unstable / crescendo angina: It occurs in less effort or rest time because of
rupture, fissuring or ulceration of atherosclerotic plaque or mural thrombus.
x. Pleural effusion: Pleural effusion is excess fluid that accumulates in the

pleura, the fluid- filled space that surrounds the lungs. It is a common manifestation of both
primary and secondary pleural diseases. Normally, not more than 15 ml of serous,
relatively acellular, clear fluid lubricates the pleural surface.
Causes of pleural effusion-
1. Increased hydrostatic pressure, e.g. Congestive Heart Failure.
2. Increased vascular permeability, e.g. Pneumonia, tuberculosis, carcinoma.
3. Decreased osmotic pressure, e.g. Nephrotic syndrome, liver cirrhosis.
4. Increased intrapleural negative pressure, e.g. Atelectasis.
5. Decreased lymphatic drainage, e.g. Mediastinal carcinomatosis.
xi. Diabetic ketoacidosis: In long standing uncontrolled or poorly

controlled diabetes mellitus, there is over production of ketone bodies which leads to the
condition termed as diabetic ketoacidosis.
Cardinal features of diabetic ketoacidosis-
1. Hyperglycaemia.
2. Hyperketonaemia/ Ketonaemia.
3. Metabolic acidosis.
Mechanism of diabetic ketoacidosis-
Lack of insulin→ ↑ lypolysis → ↑ free fatty acid in plasma → ↑ β- oxidation → ↑
Acetyl Co-A formation → ↑ formation of aceto-acetate → ↑ formation of ketone
bodies → Hyperglycaemia → Acidosis (Diabetic acidosis)
Clinical Features-
Signs: Polyuria, polydypsia, weight loss, weakness, nausea and vomiting, leg crams,
blurred vision, abdominal pain.
Symptoms: Dehydration, hypertension, tachycardia, cold extremities, air hunger,
acetone smell in breathing, confusion, drowsiness, coma.
Treatment of diabetic ketoacidosis-
1. Correction of dehydration by intravenous normal saline.
2. Short acting or soluble insulin- by infusion pump or IM.
3. Potassium replacement- if there is hypokalaemia.
4. Intravenous antibiotics- if there is infection.
xii. Oedema: 1. Oedema may be defined as, “an excessive extravascular

accumulation of fluid.”
2. Accumulation of excess fluid in the interstitial tissue space or body cavities.
Classification of oedema: I. On extent of involvement-
1. Localized oedema, e.g. pulmonary oedema, ascities,
lymphoedema, inflammatory oedema.
2. Generalized oedema, e.g. cardiac oedema, renal oedema, hepatic
oedema, nutritional oedema.
II. On Pathophysiology-
1. Inflammatory oedema
2. Non-inflammatory oedema.
III. On the nature of fluid-
1. Exudate
2. Transudate
IV. On clinical basis-
1. Pitting oedema
2. Non-pitting oedema
xiii. Malignant cell: The term literally means growing worse and resisting
treatment. It is used as a synonym for cancerous and connotes a harmful condition that
generally is life-threatening.
There are several features that can be used to differentiate normal cells from
malignant cells-
1. Invasion: Malignant cells do not respect tissue boundaries, and can be seen
infiltrating or invading into surrounding structures
2. Increased mitotic rate: Mitoses are rarely seen in normal tissues. Malignant cells
will often have increased numbers of mitoses. Mitoses are typically counted 'per high
power field'. More aggressive tumours typically have a higher mitotic rate; however these
tumours are typically more sensitive to radiation.
3. Differentiation and Anaplasia: Normal cells are usually structured in a particular
way that corresponds with their function. This is known as differentiation. Malignant cells
may become less differentiated as part of their path to malignancy. This is known as
anaplasia.
a. Well differentiated malignant cells show features similar to the parent
tissue. For example, well differentiated adenocarcinoma cells will tend to form
gland-like structures; well differentiated squamous cell carcinomas may show
intercellular bridging or keratin formation.
b. Poorly differentiated cells have lost most of their resemblance to the
parent tissue, which may be difficult to identify without special staining techniques.
c. Anaplastic cells have no resemblance to their parent tissue, and usually
indicate a very aggressive malignancy.
xiv. Shock:
a. Shock may be defined as, “widespread hypoperfusion of tissue due to
reduction in the blood volume or cardiac output or redistribution of blood
resulting in an inadequate effective circulatory volume.”
b. Shock is a disorder that results from systemic hypoperfusion due to
reduction either in cardiac output or in the effective circulatory blood
volume.
Classification of shock-
a. Cardiogenic shock, e.g. Myocardial infarction, Ventricular rupture, Arrhythmia,
Cardiac temponade, Pulmonary embolism.
b. Hypovolaemic shock, e.g. Haemorrhage, fluid loss (vomiting, diarrhoea, burns,
trauma).
c. Septic shock, e.g. overwhelming microbial infection, endotoxic shock, Gram
positive septicaemia, Fungal species, Super antigen.
d. Neurogenic shock, e.g. Anaesthetic accident, spinal injury.
e. Anaphylactic shock, e.g. Generalized IgE mediated hypersensitivity reaction
(Type-I hypersensitivity).
xv. Hyperplasia: Definition-

a. Increase in the number of cells in an organ or tissue with increase in volume is
called hyperplasia.
b. Hyperplasia is an increase in the number of cells in an organ or tissue, which then
may increase in volume.
c. Hyperplasia is an increase in the size of an organ or tissue due to an increase in
number of its specialized constituent cells.
Causes of hyperplasia: a. Induction of different genes encoding GF, GFR.

b. Increased local production of GF, GFR.
c. Results in cellular proliferation. Hormone may act as
growth factor.
Types of Hyperplasia- A. Physiological-

c. Hormonal hyperplasia, e.g. Development of female breast during
puberty or pregnancy
d. Compensatory hyperplasia, e.g. hyperplasia of liver after partial
hepatectomy.
B. Pathological-
a. Prostate- benign hyperplasia in old age
b. Male breast- gynaecomastia.
c. Endocrine glands- parathyroid hyperplasia in chronic renal failure.
xvi. Granuloma: Granuloma is a focus of chronic inflammation consisting of a

microscopic aggregation of macrophages and epithelioid cells which are modified
macrophages surrounded by a collar of mononuclear leucocytes, principally lymphocytes.
So, granuloma is defined as focal collection or aggregation of modified macrophages or
epithelioid cells. This focus of chronic inflammation may be surrounded by lymphocytes,
plasma cells, fibroblasts and giant cells particularly Langhan’s and foreign body type of
giant cells. The center may or may not contain caseation necrosis.
Types of Granuloma-
a. Depending on the presence or absence of caseation necrosis:
1. Caseous granuloma, e.g. Tuberculosis.
2. Non-caseous granuloma, e.g. Other granuloma.
b. Depending on the immune response/ Pathogenesis:
1. Immune granuloma, e.g. Tuberculosis
2. Non-immune granuloma, e.g. Foreign body granuloma and other
granuloma.
xvii. Frozen section: It is a special procedure by which a

fresh tissue is rapidly frozen, the matter within the tissue turns into ice and the tissue
becomes firm- the ice acting as embedding medium. Therefore, sections are produced
without the use of dehydrating solution, clearing agent or wax embedding.
Merits of frozen sections-
1. This is a quick diagnostic procedures. Sections are stained and prepared for
microscopic examination in about 10-15 minutes.
2. Every type of staining can be done.
3. There is minimal shrinkage of tissues as compared to paraffin sections.
4. Lipids and enzymes which are lost in routine paraffin sections can be
demonstrated.
Demerits of frozen sections-

1. It is difficult to cut serial sections.
2. It is not possible to maintain tissue blocks for future use.
3. Sections cut are thicker.
4. Structural details tend to be distorted due to lack of embedding medium.
Indication of frozen sections-
1. Diagnosis of malignancy in suspected cases.
2. To see the excision margin whether it is free or invaded by the tumour.
3. Diagnosis of Hirschprung diseases (congenital megacolon) in neonates,
children.
4. Recurrence of metastasis.
5. IHC (immunohistochemistry) of fresh sections.
xviii. Immunohistochemistry: Immunohistochemistry (IHC)

is a technique for identifying cellular or tissue constituents (antigens) by means of antigen
antibody reactions. The site of antibody binding may be identified either by direct labelling
of the primary antibody or by use of a secondary labelling antibody.
Advantages of IHC-
1. Can be applied to routinely processed material, even if stored for a long periods.
2. Provide an accurate correlation with traditional morphologic parameters.
3. Compatible with most currently used fixative.
4. IHC can be applied to decalcified material, previously stained microscopy
sections.
5. Can also be applied to cytologic preparation and to electron microscopy.
Disadvantages of IHC-
1. May give rise to false negative results.
2. Properly trained and experienced laboratory personnel are needed to interpret
the results.
xix. Decalcification: Decalcification is a process by which

bony tissue is softened and processed so that they can be cut.
Methods of processing-
1. Nitric acid method: Nitric acid - 5 ml
Small pieces of bony tissues are left in aqueous nitric acid solution for 1-5
days. The solution is preferably changed everyday and the tissue is to be tested to see if it
has become soft enough to cut. This is done by bending the tissue or by piercing a knife or
needle.
2. Formic acid method: Formic acid (SG 1.2) - 5-10 ml
Distilled water to - 100 ml.
It takes a longer time, 5-14 days for decalcification. The method is very
satisfactory where speed is not essential.
The acid from the tissue must be removed with 70% alcohol giving three
changes during 24 hours.
xx. Special stain: These are applied for demonstration of

certain substances or constituents of the cells or tissues. The staining depends upon either
physical, chemical or differential solubility of the stain with the tissue. The principles of
some of the staining procedures are well known while those of others are unknown.
The special stains and techniques (which don’t involve a stain) can be divided into
three major types-
1. Stains used for particular parts of tissues, usually components of connective
tissues which do not readily seen or well stained by the haematoxylin stain.
2. Stains for particular substances, like iron and mucin, which do not pick up
the haematoxylin stain.
3. Stains for micro-organisms such as fungi and bacteria.
The various common special stains in use in the laboratory are as under-
1. Sudan Black/ Oil Red O: These stains are used for demonstration of fat.
2. van Gieson: This stain is used for staining of collagen fibers.
3. Masson’s trichrome: This stain is used for staining of muscle.
4. Reticulin: This is used for demonstration of reticulin fibers.
5. Congo Red: This stain is used for demonstration of amyloid, an extracellular
fibrillar proteinaceous substance.
6. Periodic Acid Schiff (PAS): This stain is used for demonstration of
glycogen and mucopolysaccharides. Can also be used for fungus.
7. Methyl Violet: This is a metachromatic stain, i.e. the tissues are stained in a
colour which is different from the colour of the stain itself. It is used for
demonstration of amyloid in tissues.
8. Prussian Blue/ Perl’s Reaction: This is used for demonstration of iron.
9. Verhoeff Vangienson (VVG): This is used for demonstration of elastic
fibers.
xxi. Quality control in histopathology laboratory: Quality

control is traditionally applicable to three phases of operation-
1. The pre-analytical phase: The pre-analytical phase is related to sample collection,
transport, accession and processing. Newer models for the pre-analytical phase also include
aspects like patient satisfaction with the collection process, professional staff satisfaction
with arrangements made by the laboratory towards sample collection and transport, etc.
2. The analytical phase: The analytical phase is related to actually carrying out the
test (manual/automated) by maintaining a standard operating procedure with accuracy and
precision. The analytical part concerns the interpretation of the slide and making an
accurate diagnosis. and
3. The post-analytical phase: The post-analytical part involves the generation and
transmission of the histopathology report, storage/disposal of samples, slides and blocks
and proper retention of test results.
xxii. BEP (Benign enlargement of prostate)/ BPH (Benign prostatic

hyperplasia)/ Nodular hyperplasia: It is characterized by hyperplasia of prostatic stromal
and epithelial cells, resulting in the formation of large, fairly discrete nodules in the peri-
urethral region of the prostate. When sufficiently large, nodules compress and narrow the
urethral canal to cause partial or sometimes complete obstruction of the urethra.
Age- Over 50 years are most often between 60-70 years.
Cause- The cause is uncertain, but likely related to effects of androgens,
dihydrotestosterone, derived from testosterone via 5, α-reducaste activity, probably
mediates growth.
Morphology-
Gross:
6. Prostate is enlarged, weight becomes 3-10 times normal (60-100gms).
7. Nodules in lateral lobe may compress urethra to a slit like orifice.
8. Nodules in middle lobe may project up into floor of urethra.
9. Cross sections shows well defined nodules of various size, colour and
consistency.
10. There is no true capsule.
Microscopic:
1. Glandular, fibroblastic and muscular proliferation.
2. Gland may be small to large and cystically dilated.
3. The epithelium is thrown up into papillary buds and infolding.
4. Basement membrane is intact.
5. Glands contain corpora amylasia.
6. Aggregates of lymphocytes found within the stroma.
Complications-
1. Chronic retention
2. Acute retention
3. Cystitis
4. Pyelonephritis
5. Hydronephrosis
6. Trabeculation and diverticulum formation of the bladder.
7. Hydroureter
8. Renal failure.
“No man is free who is not master of himself.”- Epictetus

BSc Laboratory Medicine Guide

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BSc in Laboratory Medicine Part-II -1-

Recommended for further reading-

1. Practical Pathology and Microbiology

Science is organized knowledge. Wisdom is organized life. - Immanuel Kant

Preface to the 2nd Edition

B.Sc in Health Technology (Laboratory Medicine) is a new diverse

Short saying often contains much wisdom. - Sophocles

Histopathology and Cytopathology

Classification of inflammation: According to duration, inflammations are of 2

b. Chronic inflammation: It is an inflammation that persists for weeks to

Difference between acute and chronic inflammation:

Causes of hypertrophy: a. Increased functional demand.

Types of hypertrophy: A. Physiological, e.g. Muscles in athlete, Uterus in

c. Hyperplasia is an increase in the size of an organ or tissue due to an increase in

Types of Hyperplasia: A. Physiological-

Difference between hypertrophy and hyperplasia:

Classification of cell injury: A. According to severity-

Definition of Adaptation /cellular adaptation: It may be defined as, “the ability of

Types of cellular adaptation:

III. Hyperplasia- It is an increase in the number cells in an organ or tissue, usually

c. Lacerated wounds: These are caused by machinery, falls on rough surfaces

Wound healing: a. Healing or wound healing is the replacement of damaged,

Factors influencing wound healing:

4. Keloid formation- it is a raised area of scar tissue due to excessive formation of

Difference between benign and malignant tumours:

b. Hypovolaemic shock, e.g. Haemorrhage, fluid loss (vomiting, diarrhoea, burns,

Pathogenesis of septic shock:

Pathogenesis of haemorrhagic/ hypovolaemic shock:

Oedema: 1. Oedema may be defined as, “an excessive extravascular accumulation of

Difference between thrombus and embolus:

Definition of Embolus: 1. Embolus is a detached intravascular solid, liquid or

d. Foreign body embolism, e.g. bullets, silk, talc.

2. Liquid: a. Fat embolism

C. According to presence or absence of infection-

Complication of thrombosis/thrombi: The thrombi are of clinical importance for

Complications/Main effects of emboli (or, embolism) are:

Laboratory Diagnosis of a tumour (or, Cancer):

Difference between necrosis and apoptosis:

Effects of ischaemia: 1. No effect if adequate collateral circulation exists.

The pathogenesis of thrombosis: Virchow's triad describes the pathogenesis of

5. Drug and chemical agents: Alcohol, narcotics, cytotoxic drugs, poisons.

1. Cellular adaptation: a. Atrophy

Tumour markers may be-

Markers Associated cancers

ii. Apoptosis (or, necrobiosis): Apoptosis is a distinctive morphologic pattern of cell

Morphology/Morphological features of Apoptosis-

iv. Paraneoplastic syndrome: A paraneoplastic syndrome is a disease or symptom

This syndrome refers to a large group of complications and medical problems in

v. Ulcer: Ulcer is a local defect or excavation due to loss of continuity of

vi. Nomenclature of tumours:

Clinical importance of urine examination:

2. Urine: a. Bile salts

Indication of Liver function test:

4. To follow up the progress of liver damage

Ankylostoma duodenale AL (Fertilized ova) AL (unfertilized ova) Trichuris trichiura

Enterobious vermicularis Taenia spp. Hymenolepsis nana

Indication of stool culture: 1. Cholera and diarrhoeal diseases-

Contraindication of lumber puncture:

Complication of lumber puncture:

Normal findings of semen analysis (Routine examination of semen with normal

3. Consistency: Normally turbid and viscid.

Detection/estimation of protein/ albumin in urine: