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Bone marrow aspiration and trephine biopsy

Bone marrow evaluation is essential to confirm or rule out certain haematological


conditions.
Bone marrow may be obtained by (i) Bone marrow aspiration or (ii) Bone marrow biopsy
Bone Marrow Aspiration
Sites of aspiration:
1. Sternum
2. Posterior superior iliac spine
3. Iliac crest
4. Anterior superior iliac spine
5. Spinous process of lumbar vertebra
6. Upper end of tibia (infants)
The needles commonly used for the bone marrow aspiration technique are Salah needle
and Klima needle
Procedure of Bone Marrow Aspiration
a) Patient is made to lie in lateral position.
b) Skin over the posterior superior iliac spine is cleaned with iodine followed by alcohol.
c) Xylocaine is injected into the skin overlying the posterior superior iliac spine and also
into the subcutaneous tissue and the periosteum..
d) Sahlas needle along with its stylet and guard is introduced through the skin cut into
the bone with rotatory clockwise and anti clockwise movement. Needle is pushed
through the cortex into the medullary bone and the resistance given way as needle
enters the medullary cavity. The guard prevents further pushing in of the needle.
e) The stylet is withdrawn and a 10ml syringe is attached to the needle. Suction is
applied to draw 0.2ml to 0.4ml of marrow into the syringe. Then suction is stopped.
f) Needle and syringe together are withdrawn and marrow is poured onto the slides
placed at an angle of 30 degree so that the blood present in marrow is drained off.
g) Using a spreader slide, smears of marrow are made and the particles of the marrow
are carried to the end of the slide. Good marrow smear contains the marrow particles
as well as trails.
Indications for Aspiration:
(A) Red cell disorders: Megaloblastic anaemia, pure red cell aplasia, pancytopenia
(B) White cell disorders- Subleukaemic / Aleukaemic leukaemia
(C) Megakaryocytic disorders ITP and other thrombocytopenia
(D) Myeloproliferative disorders polycythaemia Vera, chronic myeloid leukaemia
(E) Myelodysplastic Syndrome
(F) Storage disorders Gauchers and Niemann Picks disease
(G) Parasitic disorders Kala azar, Falciparum malaria
(H) Plasma cell disorders Multiple myeloma
(I) For evaluation of iron stores
(J) Metastatic tumour deposits

Indications for biopsy:


1. Aplastic anaemia
2. Myelofibrosis
3. Storage disorders
4. Metastatic deposits
5. Miliary tuberculosis
Observation:

Cellularity
Erythropoiesis
Myelopoiesis
Megakaryocytes
M;E ratio
Plasma cells
Abnormal cells

Stains used on the smears:


Romanawsky stain
Perls stain for iron
Immunohistochemistry stain

Breast Cancer
Introduction
Types
Aetiopathogenesis
hereditary breast cancer
sporadic breast cancer
Mechanism of carcinogenesis
Classification
carcinoma in situ
o Ductal carcinoma in situ (DCIS; Intraductal carcinoma)
o Lobular carcinoma in situ (LCIS)
invasive (infiltrating) carcinoma
o Invasive (infiltrating) ductal carcinoma
o invasive (infiltrating) lobular carcinoma
o Paget disease of nipple
o Medullary carcinoma
Metastasis
Major Prognostic factors
Minor prognostic factors
Treatment

Introduction
one of the most common malignant tumour of the women
3% of breast cancers are hereditary, constituting 25% of familial cancers
Types
1. Sporadic breast cancer
2. Hereditary breast cancer
Aetiology & Pathogenesis
Sporadic Breast Cancer possibly due to hormonal
Metabolites of estrogen can cause mutations or generate DNA-damaging
free radicals
via its hormonal actions, estrogens drive the proliferation of premalignant
lesions as well as cancers.
Major risk factors:
elderly age (> 50 Yrs)
gender (F>M)
early menarche (<11 Yrs) and late menopause
nulliparous (pregnancy results in terminal differentiation of epithelial cells,
removing them from the potential pool of cancer precursors)
exogenous estrogen administration
postmenopausal obese women - due to synthesis of estrogens in fat depots
overexpression of ER
Radiation Exposure - exposer to therapeutic radiation or atom bomb exposure
have a higher rate of breast cancer (Modern mammographic screening uses
low doses of radiation and is unlikely to have an effect on the risk of breast
cancer)
Geographic Influence - higher in the United States and Europe
Race lower in African-American ancestry, highest rates in Caucasian
increased dietary fat, moderate or heavy alcohol consumption
carcinoma of contra lateral breast
carcinoma of endometrium
atypical hyperplasia in breast biopsies
Lower risk:
Exercise
Breast-Feeding (longer the women breast-feed, the greater is the reduction in
the risk)
Tabacco is not associated

Hereditary Breast Cancer associated with family history or germ-line mutations

Associated with
BRCA1 and BRCA2 - highly penetrant autosomal dominant genes
multiple affected first-degree relatives
early occurrence i.e., before menopause (20 years younger than sporadic)
multiple cancers (colon, prostate, and pancreas)
male breast cancer(BRCA2)
family members also develop ovarian cancer
high risk of developing ovarian carcinoma (BRCA1)
Features of BRCA1-associated breast cancers
poorly differentiated (syncytial growth pattern with pushing margins & have a
lymphocytic response)
do not express hormone receptors
do not overexpress HER2/neu (an epidermal growth factor receptor that is
commonly overexpressed in breast cancer)
(BRCA2-associated breast carcinomas do not have distinct features)
Other gene mutations
Checkpoint kinase gene (CHEK2)
o important component of the recognition and repair of DNA damage
Li-Fraumeni syndrome (due to a germ-line mutation in the p53 gene)
p53 gene
Cowden syndrome ("multiple hamartoma syndrome" due to a mutation of the
PTEN gene on chromosome 10q)
PTEN gene
Peutz-Jeghers syndrome (mutations in the LKBI gene)
ATM gene (ataxia telangiectasia mutation)

at least two-thirds of familial risk unexplained

Mechanisms of Carcinogenesis
A general model for carcinogenesis is that a normal cell must achieve seven
new capabilities to become malignant
1. evasion of growth-inhibiting signals
2. evasion of apoptosis
3. self-sufficiency in growth signals
4. genetic instability
5. limitless replicative potential
6. increased angiogenesis
7. ability to invade

each of the new capabilities can be achieved by a change in one of many


genes
o e.g., changes in ER, EGF-R, RAS, or HER2/neu may result in selfsufficiency in growth signals
one cellular alteration (e.g., p53) can affect more than one of these
capabilities

Early stage (Proliferative changes)


related to evasion of growth-inhibiting signals, evasion of apoptosis, and selfsufficiency in growth signals
there is abnormal expression of hormone receptors and abnormal regulation
of proliferation in association with hormone receptor positivity
Later stage (atypical hyperplasia)
due to genetic instability, in the form of LOH
Last stage (Carcinoma in situ)
nuclear enlargement, irregularity, and hyperchromasia (aneuploidy)
o due to limitless replicative potential
increased angiogenesis
o due to direct stimulation by the malignant cells, secondary stimulatory
effects on stromal cells, or the loss of inhibition of angiogenesis by
myoepithelial cells
Final stage (Carcinoma in situ to invasive carcinoma)
primarily due to the loss of the basement membrane & tissue integrity caused
by the abnormal function of myoepithelial and stromal cells

Classification of Breast cancer

majority are adenocarcinoma


< 5% other types (i.e., squamous cell carcinomas, phyllodes tumors,
sarcomas, and lymphomas)

Carcinomas
divided in to in situ carcinomas & invasive carcinomas
Carcinoma in situ
a neoplastic population of cells limited to ducts and lobules by the basement
membrane
does not invade into lymphatics and blood vessels
cannot metastasize
classified as ductal carcinoma in situ (DCIS) or lobular carcinoma in situ
(LCIS) on the basis of the resemblance of the involved spaces to ducts and
lobules
Invasive carcinoma (syn "infiltrating" carcinoma)
has invaded beyond the basement membrane into stroma
can invade into the vasculature
cause regional lymph node metastasis
cause distant sites
types - Invasive ductal carcinoma and Invasive lobular carcinoma
All carcinomas arise from the terminal duct lobular unit, and the terms
"ductal" and "lobular" do not imply a site or cell type of origin
Ductal Carcinoma in Situ (DCIS; Intraductal Carcinoma)
consists of a malignant population of cells limited to ducts by the basement
membrane
myoepithelial cells are preserved
clonal proliferation involving only a single ductal system
cells can spread throughout ducts and lobules and produce extensive lesions
clinically cannot be detected by inspection or palpation
shows calcifications on mammography
progress to invasive carcinoma
proper diagnosis & appropriate therapy is important
associated with recurrence
Risk factors for recurrence
1. high grade
2. larger size
3. ill defined margins
death rate is < 2%

Morphology (five architectural subtypes; single pattern or mixed pattern)


1. Comedocarcinoma
solid sheets of pleomorphic cells with high-grade nuclei and central necrosis
Noncomedo DCIS
consists of a monomorphic population of cells with nuclear grades ranging
from low to high
2. Cribriform DCIS shows intraepithelial spaces evenly distributed and regular in
shape
3. Solid DCIS completely fills the involved spaces
4. Papillary DCIS grows into spaces and lines fibrovascular cores typically
lacking the normal myoepithelial cell layer
5. Micropapillary DCIS is recognized by bulbous protrusions without a
fibrovascular core

Paget disease of the nipple


Introduction
rare manifestation of breast cancer (1% to 2% of cases)
characterised by presence of malignant cells in the epidermis of nipple
Mechanism
Malignant cells (Paget cells) extend within the ductal system into nipple skin
without crossing the basement membrane
Clinical features
presents as a unilateral erythematous eruption with a scale crust of a nipple
pruritus is common and might be mistaken for eczema
palpable mass is present in 50% to 60%
Morphology
Paget cells are large containing moderate amount of cytoplasm and
hyperchromatic nuclei. They are arranged single or in tiny groups situated in
the epidermal layer.
overexpress HER2/neu
Clinical significance
almost all will have an underlying invasive carcinoma
Prognosis
depends on the extent of the underlying carcinoma

Lobular Carcinoma in Situ (LCIS)


consists of a malignant population of cells limited to lobules by the basement
membrane
always an incidental finding
not associated with calcifications
bilaterality & multicentricity more common
more common in young women
lack expression of e-cadherin,
have the same genetic changes as an adjacent area of invasive carcinoma
(e.g., LOH on 16q, the site of the gene for e-cadherin)
progress to invasive carcinomas
treatment
o bilateral prophylactic mastectomy
o tamoxifen therapy
o clinical follow-up and mammographic screening
Morphology
Similar to invasive lobular carcinoma
consist of small cells that have oval or
round nuclei with small nucleoli that do
not adhere to one another
signet-ring cells containing mucin are
present commonly
involved acini remain recognizable as
lobules
expresses ER and PR

Invasive (Infiltrating) Carcinoma

presents as a palpable mass


have axillary lymph node metastases
may be fixed to the chest wall or cause dimpling of the skin
lymphatics may become so involved as to block the local area of skin
drainage and cause lymphedema and thickening of the skin, a change
referred to as peau d'orange
tethering of the skin to the breast by Cooper ligaments mimics the
appearance of an orange peel
retraction of the nipple may develop, when the tumor involves the central
portion of the breast
mammography shows a density
"inflammatory carcinoma" refers to the clinical presentation of a carcinoma
extensively involving dermal lymphatics, resulting in an enlarged
erythematous breast. The underlying carcinoma usually has a diffuse
infiltrative pattern and typically does not form a discrete palpable mass. This
can result in confusion with inflammatory conditions and delay in diagnosis.
The diagnosis is made on clinical grounds and does not correlate with a
specific histologic type of carcinoma

Invasive Carcinoma, No Special Type (Invasive Ductal Carcinoma)


Invasive carcinomas of no special type include the majority of carcinomas
(70% to 80%) that cannot be classified as any other subtype.
Macroscopy

most carcinomas are firm to


hard and have an irregular
border
Within the center of the
carcinoma, there are small
pinpoint foci or streaks of chalky
white elastotic stroma and
occasionally
small
foci
of
calcification
characteristic grating sound (similar to cutting a water chestnut) when cut or
scraped

Microscopy
well differentiated, moderately differentiated or poorly differentiated
Well-differentiated tumors
o consist of tubules lined by minimally atypical cells
o express hormone receptors and do not overexpress HER2/neu.
poorly differentiated
o composed of anastomosing sheets of pleomorphic cells
o less likely to express hormone receptors & more likely to overexpress
HER2/neu
most carcinomas induce a marked increase in dense, fibrous desmoplastic
stroma, giving the tumor a hard consistency on palpation and replace fat,
resulting in a mammographic density (scirrhous carcinoma)

Invasive Lobular Carcinoma

present as a palpable mass or mammographic density


produce only a vaguely thickened area of the breast or subtle architectural
changes on mammography
metastases can also be difficult to detect clinically and radiologically owing to
this type of invasion
have a greater incidence of bilaterality
increasing among postmenopausal women - may be related to the use of
postmenopausal hormone replacement therapy
Well-differentiated and moderately differentiated carcinomas
o diploid, express hormone receptors, and are associated with LCIS
o HER2/neu overexpression is very rare
Poorly differentiated lobular carcinomas
o aneuploid, often lack hormone receptors, and may overexpress
HER2/neu
o lobular carcinomas have the same prognosis as carcinomas of NST
show a loss of a region on chromosome 16 (16q22.1) that includes a cluster
of at least eight genes responsible for cell adhesion, including e-cadherin and
-catenin
Metastases to the peritoneum and retroperitoneum, the leptomeninges
(carcinomatous meningitis), the gastrointestinal tract, and the ovaries and
uterus are more frequently observed

Morphology
Gross
firm to hard with an irregular margin
Micro:
hallmark is the pattern of single
infiltrating tumor cells often only one
cell in width (Indian file)
desmoplastic response may be
minimal or absent
cells have the same cytologic
features as LCIS and lack cohesion,
without formation of tubules or
papillae
signet-ring cells are common
cells are arranged in concentric rings
surrounding normal ducts

Metastasis
lymphnode
lungs
bones
liver
adrenals
brain
meninges
Prognostic Factors
Major prognostic factors
o insitu / invasive
o distant metastasis
o lymphnode metastasis
o tumour size
o local invasion
1. Invasive carcinoma or in situ disease
in situ carcinoma cannot metastasize associated with better prognosis.
invasive carcinomas associated with poor prognosis
2. Distant metastases - associated with poor prognosis
3. Lymph node metastases
most important prognostic factor in the absence of distant metastases
o No nodes - 70% to 80% (10 Yrs survival)
o 1 to 3 nodes 35% to 40%
o > 10 nodes 10% to 15%
4. Tumour size
second most important prognostic factor
node-negative carcinomas under 1 cm in diameter have best prognosis
10-year survival is approximately 90%
5. Local invasion
into skin or skeletal muscle are associated with distant disease
6. Inflammatory carcinoma
poor prognosis
Minor Prognostic Factors
o histological subtype
o tumour grade
o ER/PR receptor
o HER2/neu
o lymphovascular invasion
o proliferative rate
o DNA content

minor prognostic factors can be used to decide among chemotherapy


regimens and/or hormonal therapies

Three of these factorsestrogen receptor, progesterone receptor, and


HER2/neuare most useful as predictive factors for response to specific
therapeutic agents.
Histologic subtypes
Special types of invasive carcinomas (tubular, mucinous, medullary, lobular, and
papillary) has better prognosis than cancers of no special type
Tumor grade
well-differentiated grade I tumors better than moderately differentiated grade II
tumors, and poorly differentiated grade III tumors
Estrogen and progesterone receptors
hormone receptor-positive cancers have a slightly better prognosis
HER2/neu. HER2 (human epidermal growth factor receptor 2 or c-erb B2 or neu)
- transmembrane glycoprotein involved in cell growth control
- overexpression associated with poor prognosis
- Trastuzumab (Herceptin) is a humanized monoclonal antibody to HER2/neu
developed to specifically target tumour cells.
Lymphovascular invasion (LVI)
strongly associated with the presence of lymph node metastases and is a poor
prognostic factor
Proliferative rate
high proliferation rates have a worse prognosis
DNA content
aneuploid tumours have a worse prognosis
Treatment
Mastectomy
Radiation
Chemotherapy
Hormone therapy
New strategies - (by pharmacologic agents or specific antibodies) of
membrane-bound growth factor receptors (e.g., HER2/neu), stromal
proteases, and angiogenesis

Medullary Carcinoma of Breast


well-circumscribed mass with a pushing (noninfiltrative) border
clinically and radiologically mistaken for a fibroadenoma
history of rapid, almost explosive, growth
syncytial growth pattern and pushing borders may reflect retention or
overexpression of adhesion molecules that could potentially limit metastatic
potential
hypermethylation of the BRCA1 promoter is observed in 67%
marked lymphoplasmacytic infiltrate surrounding the tumor
lymphatic or vascular invasion is never seen
lymph node metastases are infrequent
HER2/neu overexpression is not observed
slightly better prognosis

STROMAL TUMORS
There are two types of stroma in the breast, intralobular and interlobular.
Biphasic tumors arise in the interlobular stroma
o fibroadenoma
o phyllodes tumor
Fibroadenoma
Intro:

most common benign tumor arise in the interlobular stroma


Clinical features:
< 30 yrs
frequently multiple and bilateral
palpable and mobile mass (mouse breast) with a mammographic density
regression usually occurs after menopause
women receiving cyclosporin A (after renal transplantation) develop
fibroadenomas
o due to drug-related growth stimulation
stroma often hyalinized and may calcify

Macroscopy
spherical nodules
well-circumscribed
1 cm to large size
C/S solid, grayish white nodules
and contain slit like spaces

Micro:
stroma is cellular, and often
myxoid, resembling intralobular
stroma
glandular and cystic spaces lined
by epithelium
epithelium may be surrounded by
stroma or compressed and
distorted by it
border is sharply delimited from
the surrounding tissue

Phyllodes Tumour (Syn. cystosarcoma phyllodes)

Introduction:
term "phyllodes tumor" is preferred, as the majority of these tumors behave
in a relatively benign fashion, and most are not cystic.
Origin:
tumour arises from intralobular stroma
6th decade
Clinical Features:
palpable masses
Macroscopy:
vary in size (few centimeters to massive lesions involving the entire breast)
C/S shows bulbous protrusions ("leaflike") into the cystic spaces
Microscopy:
Stroma high cellularity, mitotic rate, nuclear pleomorphism
Epithelium covering the stroma and extending into the cystic spaces
Borders infiltrative

Increased stromal cellularity, cytologic atypia, and stromal


overgrowth, giving rise to the typical leaflike architecture.

Clinical significance:
must be excised with wide margins or by mastectomy
majority are low-grade; recur locally
minority high-grade; recur locally and metastases by haematogenous
o only the stromal component metastasizes
axillary lymph node dissection is not indicated

Casts
Introduction:
Urinary casts are formed in the distal convoluted tubule (DCT) or the collecting duct.
The proximal convoluted tubule (PCT) and loop of Henle are not locations for cast formation.
Hyaline casts are composed primarily of a mucoprotein (Tamm-Horsfall protein) secreted by
tubular cells.

Even with glomerular injury causing increased glomerular permeability to plasma


proteins with resulting proteinuria, most matrix or "glue" that cements urinary casts
together is Tamm-Horsfall mucoprotein, although albumin and some globulins are also
incorporated.
Factors which favour cast formation actually favour protein denaturation and
precipitation.
low flow rate
high salt concentration
low pH
Significance:
Hyaline casts can be seen even in healthy patients
RBC casts - glomerulonephritis, with leakage of RBC's from glomeruli, or severe
tubular damage
WBC casts - acute pyelonephritis
Granular and waxy casts - derive from renal tubular cell casts. When cellular casts
remain in the nephron for some time before they are flushed into the bladder
urine, the cells may degenerate to become a granular cast, and ultimately, a waxy
cast
Broad casts - originate from damaged and dilated tubules and are therefore seen in
end-stage chronic renal disease
Telescoped urinary sediment
is one in which red cells, white cells, oval fat bodies, and all types of casts are
found in more or less equal profusion
Occurs in 1) lupus nephritis 2) malignant hypertension 3) diabetic
glomerulosclerosis, and 4) rapidly progressive glomerulonephritis

Coombs Test (Anti globin Test)

to detect incomplete antibody

Coombs serum
Broad spectrum Anti IgG + Anti C
Specific
Anti IgG / Anti IgM / Anti C
Types of Coombs Test
Direct Antiglobulin Test (DAT)
to find out whether the patients RBCs are coated with incomplete antibodies
Indirect Antiglobulin Test (IAT)
to find out whether incomplete antibodies are present in the patients serum
Result
Agglutination positive; No agglutination negative
Method:
DAT
add 2 drops of 3% RBC suspension
take 2 drops of Coombs serum
incubate at 37C X 1 min
centrifuge at 250g C 1 min
read the results microscopically
IAT

2 drops of 3% RBC + 2 drops of Pts serum


incubate at 37C X 1 h
wash the cells X 4 time
add 2 drops of Coombs serum
continue as for DAT

IAT - applications
1. screening the sera as a means of antibody detection
2. characterising alloantibodies in typing unknown cells
3. as a cross matching procedure

Cytology
Introduction:
Cyto = cell; Logy = study
It is an important diagnostic tool and is a sub specialty of pathology.
Division:
1. Exfoliative cytology
2. Fine Needle Aspiration Cytology (FNAC)
1. Exfoliative cytology
It deals with cells exfoliated from the surface. They are obtained by scraping the surface
or aspirating the fluid
Papsmear cells exfoliated from cervix
Sputum cells exfoliated from bronchopulmonary tree
Gastric lavage Stomach
Pleural, Ascitic, CSF
2. FNAC:
Cells are obtained by aspirating the lesion using a fine needle (23G) and a 10 ml syringe
e.g.,
palpable lesions - breast, thyroid, lymphnode etc.
deep - lung, pancreas, liver, prostate, kidney etc.
Procedure:
Cells are made into smears on the slides
Smears are either air dried or fixed in alcohol
They are stained by papstain or Romanawsky stain and are examined under the
microscope.
Application:
diagnosis of early cancer
female genital tract especially the cervix
respiratory tract especially the lung
genitourinary tract
diagnosis of lumps
palpable lesions - breast, thyroid, lymphnode etc.
deep - lung, pancreas, liver, prostate, kidney etc.
diagnosis of recurrent tumours / metastasis
population screening
Advantages:
outpatient procedure, does not require hospitalisation
does not require anaesthesia
rapid diagnosis
economical
less painful
Limitations:
less sensitive than histopathology
tissue architecture is not preserved
false negative occurs, if the needle did not hit the target.

DIC Lab Investigations


Introduction:
DIC means disseminated intravascular coagulation. i.e., coagulation occurs inside the
vessels (especially the micro vessels) throughout the body.
Principle:
Consumption of all the coagulation factors and inhibitors. So their levels got
decreased.
Platelets are also utilized in the coagulation and so reduced in number.
Coagulation forms fibrin mesh inside the capillaries and the RBCs has to squeeze
thro it and get destroyed (haemolysed).
At the same time, wide spread fibrinolysis also will take place. That leads to
formation of fibrin degradation products.
Investigations:
Screening tests:
1. Prothrombin time Increased.
2. aPTT - Increased..
3. Thrombin Time Increased.
4. Platelet count Decreased.
Elaborate Tests:
1. Detection of FDP (Fragment D, Fragment E), by latex agglutination test Positive.
2. Test for fibrin monomer by protamine sulphate test Positive.
3. Quantitative assays for Factor I, V and VIIIc Decreased.
4. Plasma antithrombin III assay Decreased.

Erythrocyte indices
(Absolute values or Wintrobes constant)
MCV (Mean corpuscular volume)
means volume of individual RBC
MCV =
PCV in Litre / Litre
RBC count / Litre
Short cut formula =

PCVX10
RBC in millions

Expressed in femtolitres
Normal value is 85 8 fl (77 93fl)
MCV (Mean corpuscular haemoglobin)
MCV=
Hb gm / Litre
RBC count / Litre
Short cut formula =

HbX10
RBC in millions

Expressed in picograms (micro micrograms)


Normal value is 29.5 2.5 pg (27-32 pg)
MCHC (Mean corpuscular haemoglobin concentration)
MCHC = Hb in gm /dl
PCV L/L
Or
MCHC = Hb X 100
PCV %
Expressed in percentage
Normal value is 34.5 1.5% (33-36%)
Significance:
The indices will give clue regarding type & aetiology of anaemia.
Iron deficiency anaemia MCV, MCH, MCHC decreased
Megaloblastic anaemia - MCV, MCH Increased; MCHC Normal / decreased

ESR
Definition:
is a rate at which the erythrocytes are getting sedimented
expressed as mm at first hour
Factors
specific gravity of RBCs
viscosity of plasma
difference between them
verticality of tube
bore of the tube
Specific gravity of RBCs is the most important factor.
directly proportional to rouleux formation
rouleux
fibrinogen
acute phase reactants (e.g., CRP)
immunoglobulin; globulin
rouleux
albumin
poikilocytosis (iron deficiency, sickle cell disease)
spherocytes (spherocytosis; artefact)
Stages of ESR:
1. Stage of rouleux formation (10 min)
2. Stage of settling (40 min)
3. Stage of packing (10 min)
Clinical significance
ESR has prognostic value rather than diagnostic.
ESR
chronic inflammation (TB, rheumatoid arthritis)
pregnancy
myocardial infarction
anaemia
multiple myeloma

ESR
polycythaemia
congestive cardiac failure
hypofibrinogenaemia
sickle cell disease
spherocytosis
Methods
Westergrens method
Wintrobe method
MicroESR method
Automated method

Frozen Section
Principle:
when tissue is frozen, the water within the tissue turns to ice and in this state the
tissue is firm and the sections can be cut easily.
lesser the temperature, harder the tissue
Application:
on table diagnosis (about 15 min)
confirmation of malignancy, clearance of tumour margin
enzyme histochemistry
acetyl cholinesterase in Hirschprung disease, ATPase in muscle biopsy
non-enzyme histochemistry
lipid, some carbohydrates
immunohistochemistry
immunofluorescent staining
silver staining in neuropathology
Method: (-15C to -20C)
1. Cryostat
2. Freezing microtome
Technique:
Preparation of tissue either unfixed or fixed tissue. Rapid freezing by liquid
nitrogen or CO2 gas or aerosol spray
Cutting the sections require skill
Staining is also rapid, as there is no need for hydrating the sections.
Disadvantages:
expensive equipment
section cutting needs a well experienced technician
interpretation also needs a well experienced pathologist
false negative result
only small sample can be processed.

LE Cell
Introduction:
LE cell means Lupus Erythematosus cell.
LE cell preparation is a test to detect the presence of antinuclear protein antibody in the
patients serum (LE factor)
Positive in
SLE (75% of the cases) and also rarely in
lupoid hepatitis
drug reaction
rheumatoid arthritis
Principle:
LE factor lyses the neutrophil nucleus in vitro.
active neutrophils will phagocytose the lysed nucleus
Essential:
LE factor
nuclear protein material (i.e., traumatised WBC)
complement
actively phagocytic neutrophil
37C
Appearance:
LE cell is a neutrophil containing LE body which is round, structureless, opaque,
homogenous pale blue mass. The nucleus of the neutrophil will be pushed to the
periphery.

Advantages:
cost effective
simple technique.
Limitation:
Less sensitive than the serum estimation of anti nuclear antibodies.
Tart cell will be confused with LE cell.

Leukaemoid reaction
Definition:
It is a non leukaemic condition which microscopically resembles leukaemia
Types:
myeloid leukaemoid reaction, resembles CML
lymphoid leukaemoid reaction, resembles CLL.
Characteristics:
Total WBC count will be markedly elevated.
presence of immature WBCs i.e., metamyelocytes, myelocytes & promyelocytes.
Conditions:
infections
severe bacterial infection, disseminated TB, pertussis, hepatitis
malignancies
Hodgkin lymphoma
gastric, breast, lung cancers
intoxications
Differentiation from leukaemia:
blast will never occur in leukaemoid reaction. blasts will be present in leukaemia
and their number vary depends upon the type.
Neutrophil alkaline phosphatase (NAP) activity Increased in leukaemoid
reaction whereas decreased in leukaemia.

Leukocyte Cytochemistry
Introduction:
Leukocyte cytochemistry encompasses the techniques used to identify diagnostically
useful enzymes or other substances in the cytoplasm of haemopoietic cells.
These techniques are particularly useful for the characterization of immature cells in
the AMLs and the identification of maturation abnormalities in the myelodysplastic
syndromes and myeloproliferative disorders.
The use of cytochemistry to characterise lymphoproliferative disorders has been
largely superseded by immunological techniques
The results of cytochemical tests should always be interpreted in relation to
Romanowsky stains and immunological techniques.
Principal uses of cytochemistry:
1. Myeloperoxidase (MPO) Positive in AML with maturation; Negative in more
primitive myeloblast. (Brown)
2. Sudan Black same as MPO. (Black)
3. PAS Block positivity in ALL, AML M6 (Erythroblasts in erythroleukaemia) (Red)
4. Esterases ANAE (-Naphthyl Acetate Esterase) and ANBE (-Naphtyl Butyrate
Esterase) Monocytic series (Brown)
5. Neutrophilic alkaline phosphatase (NAP) scores is low in chronic phase of CML;
high in leukaemoid reaction (Blue)
6. Pearls reaction - demonstration of ring sideroblasts in MDS (Blue)
7. Tartrate-Resistant Acid Phosphatase (TRAP) positive in hairy cell leukaemia
(Brown)

Packed Cell Volume (PCV) / Haematocrit


Equipments
Wintrobes tube & Pasteur pipette
Procedure
1. Using the Pasteur pipette, fill the Wintrobes tube to 0 mark
2. Centrifuge at 2000 to 2300g for 30 minutes
3. After centrifugation, layers are noted in the Wintrobe tube as under
a. Uppermost layer of plasma
b. Thin white layer of platelets
c. Greyish pink layer of WBCs
d. Lowermost red column of RBCs
4. Note the lowermost height of column of packed RBC and express it in percentage
Normal Values
Men
: 40-55 %
Women
: 36-48 %
Note: Grey white layer of WBCs and platelets interposed between plasma and RBC
column is called buffy coat.
Uses of PCV
to diagnose anaemia or polycythaemia
Other methods to detect PCV
Microhaematocrit method.
Uses of buffy coat
to screen for microfilaria larvae
to find out the blasts in subleukaemic leukaemia
to detect LE cell

Reticulocyte Count
Introduction:
Reticulocytes are juvenile red cells
they contain remnants of the ribosomal ribonucleic acid (RNA)
Principle:
RNA reacts with basic dye, brilliant cresyl blue, or New methylene blue to form a
blue or purple precipitate of granules or filaments.
This reaction takes place only in vitally stained unfixed preparations
Procedure:
Equal mixture of 1% dye mixed with blood and incubated at 37C for 15 minutes
Then smears are made
Observed under oil immersion.
Count 1000 consecutive RBC and express the reticulocyte in percentage.
Appearance:
Reticulocyte appears bigger than the mature RBC and contains blue precipitates in the
form of reticulum (net work) or granules.
Normal count:
0.2 to 2 %
Corrected Reticulocyte count:
= Observed Retic count X

Measured PCV or Hb
Appropriate Normal PCV or Hb

Significance:
The number of reticulocytes in the peripheral blood is a fairly accurate reflection of
erythropoietic activity.
Increased Haemolytic anaemia, Nutritional
anaemia responding to the treatment.
Decreased or Absent Aplastic anaemia,
Aplastic crisis in haemolytic anaemias.

Laboratory Diagnosis of Cancer


Methods:
1. Histopathological examination routinely used method in surgical pathology
services.
Excision biopsy
Incision biopsy
Surgical removal of diseased organs
2. Cytology rapid diagnostic procedure, simple, but less sensitive than histopathology
FNAC
Papsmear
Fluid cytology
3. Frozen section rapid diagnostic procedure, requires expertise in making the sections
and also in the interpretation.
4. Immunohistochemistry an adjuvant technique to detect the tumour marker in the
surgical pathology services. It is very expensive and has got its own limitations.
5. Flow Cytometry used to find out the DNA ploidy and other markers. It is applied
only in certain tumour like haematological malignancies.
6. Electron microscopy used to find out the exact cell lineage of a tumour, by
detecting the ultramicroscopic structure. It requires very costly equipment.
7. Molecular diagnosis recently developed technique.
PCR
FISH (Fluorescent In Situ Hybridization)
DNA-microarray analysis

Tumour Markers
Introduction:
A tumour marker is a substance found in the blood, urine, or body tissues that may be
elevated in cancer.
They are used in oncology to help detect the presence of cancer.
Production:
Tumour markers can be produced directly by the tumour or by non-tumour cells as a
response to the presence of a tumour.
Classification
Tumour markers can be classified in two groups: Cancer-specific markers and tissuespecific markers.
Cancer-specific markers
Cancer-specific markers are related to the presence of certain cancerous tissue. Because
there is a large overlap between the many different tumour tissue types and the markers
produced, these markers might not be specific in making a diagnosis. They can, however,
be useful in the follow-up of treated patients to describe progress of the disease or
response to treatment.
CEA, or carcinoembryonic antigen
first noted to be produced by tumours of the gastrointestinal system
it was also produced by the occasional lung and breast cancer
an elevated level does not necessarily mean a bowel cancer. However, in a patient
with a history of a treated bowel cancer, a rising CEA level can be an early sign of
recurring bowel cancer.
Tissue-specific markers
related to specific tissues which have developed cancer
not specifically related to the tumour, and may be present at elevated levels when no
cancer is present
unlike the previous group, elevated levels point to a specific tissue being at fault.
o elevated PSA Ca prostate, hyperplasia or trauma to prostate
o elevated beta-HCG choriocarcinoma, hydatidiform mole, pregnancy
o elevated AFP liver cancer
Application:
* Screening for common cancers on a population basis
e.g., elevated prostate specific antigen suggests prostate cancer.
* Monitoring of cancer survivors after treatment
e.g., elevated AFP in a previously treated for endodermal sinus tumour suggests relapse.
* Diagnosis of specific tumour types, particularly in certain brain tumours and other
instances where biopsy is not feasible.

Limitations:
An elevated level of a tumour marker can indicate cancer; however, there can also be
other causes of the elevation. Hence, tissue diagnosis (biopsy & histopathological
examination) is required for confirmation.
Method of detection:
1. Biochemical method to detect the tumour markers in the blood and body fluids
2. Immunohistochemitry method to detect the tumour markers in the tissue.

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