misc answers

8/19/2019 Misc Answers

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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. Sternum2. Posterior superior iliac spine3. Iliac crest4. Anterior superior iliac spine5. Spinous process of lumbar vertebra6. 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) Sahla’s needle along with its stylet and guard is introduced through the skin cut intothe bone with rotatory clockwise and anti clockwise movement. Needle is pushedthrough 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e) The stylet is withdrawn and a 10ml syringe is attached to the needle.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 Then suction is stopped.f) Needle and syringe together are withdrawn and marrow is poured onto the slides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 – Gaucher’s and Niemann Pick’s disease(G) Parasitic disorders – Kala azar, Falciparum malaria (H) Plasma cell disorders – Multiple myeloma(I) For evaluation of iron stores(J) Metastatic tumour deposits


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Indications for biopsy:

1. Aplastic anaemia2. 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


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Breast CancerIntroductionTypes

Aetiopathogenesis

• hereditary breast cancer

•

sporadic breast cancerMechanism of carcinogenesisClassification

• carcinoma in situo Ductal carcinoma in situ (DCIS; Intraductal carcinoma)o Lobular carcinoma in situ (LCIS)

• invasive (infiltrating) carcinomao Invasive (infiltrating) ductal carcinomao invasive (infiltrating) lobular carcinomao Paget disease of nippleo Medullary carcinoma

• Metastasis• Major Prognostic factors

• Minor prognostic factors

• Treatment


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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 cancer2. Hereditary breast cancer

Aetio logy & PathogenesisSporadic Breast Cancer – possibly due to hormonal

• Metabolites of estrogen can cause mutations or generate DNA-damagingfree radicals

• via its hormonal actions, estrogens drive the proliferation of premalignantlesions as well as cancers.

Major risk factors:

• elderly age (> 50 Yrs)

• gender (F>M)• early menarche (


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

o e.g., changes in ER, EGF-R, RAS, or HER2/neu may result in self-sufficiency 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 self-sufficiency in growth signals

• there is abnormal expression of hormone receptors and abnormal regulationof proliferation in association with hormone receptor positivity

Later stage (atypical hyperplasia)

• due to genetic instability, in the form of LOHLast stage (Carcinoma in situ)

• nuclear enlargement, irregularity, and hyperchromasia (aneuploidy)o due to limitless replicative potential

• increased angiogenesiso due to direct stimulation by the malignant cells, secondary stimulatory

effects on stromal cells, or the loss of inhibition of angiogenesis by

myoepithelial cellsFinal stage (Carcinoma in situ to invasive carcinoma)

• primarily due to the loss of the basement membrane & tissue integrity causedby the abnormal function of myoepithelial and stromal cells


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Classif ication 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 basementmembrane

• 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 grade2. larger size3. ill defined margins

• death rate is < 2%


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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 rangingfrom low to high

2. Cribriform DCIS shows intraepithelial spaces evenly distributed and regular inshape

3. Solid DCIS completely fills the involved spaces4. Papillary DCIS grows into spaces and lines fibrovascular cores typically

lacking the normal myoepithelial cell layer5. Micropapillary DCIS is recognized by bulbous protrusions without a

fibrovascular core


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Paget disease of the nippleIntroduction

• rare manifestation of breast cancer (1% to 2% of cases)

• characterised by presence of malignant cells in the epidermis of nippleMechanism

• Malignant cells (Paget cells) extend within the ductal system into nipple skinwithout 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 andhyperchromatic nuclei. They are arranged single or in tiny groups situated inthe epidermal layer.

• overexpress HER2/neu

Clinical significance• almost all will have an underlying invasive carcinomaPrognosis

• depends on the extent of the underlying carcinoma


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Lobular Carcinoma in Situ (LCIS)

• consists of a malignant population of cells limited to lobules by the basementmembrane

• 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

• treatmento bilateral prophylactic mastectomyo tamoxifen therapyo clinical follow-up and mammographic screening

Morphology Similar to invasive lobular carcinoma

• consist of small cells that have oval orround nuclei with small nucleoli that donot adhere to one another

• signet-ring cells containing mucin arepresent commonly

• involved acini remain recognizable aslobules

• expresses ER and PR


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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 changereferred to as peau d'orange

• tethering of the skin to the breast by Cooper ligaments mimics theappearance of an orange peel

• retraction of the nipple may develop, when the tumor involves the centralportion of the breast

• mammography shows a density

• "inflammatory carcinoma" refers to the clinical presentation of a carcinomaextensively involving dermal lymphatics, resulting in an enlargederythematous breast. The underlying carcinoma usually has a diffuseinfiltrative 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 aspecific 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 irregularborder

• Within the center of thecarcinoma, there are smallpinpoint foci or streaks of chalkywhite elastotic stroma andoccasionally small foci ofcalcification

• characteristic grating sound (similar to cutting a water chestnut) when cut orscraped


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Microscopy

• well differentiated, moderately differentiated or poorly differentiated

• Well-differentiated tumorso consist of tubules lined by minimally atypical cellso express hormone receptors and do not overexpress HER2/neu.

• poorly differentiated

o composed of anastomosing sheets of pleomorphic cellso less likely to express hormone receptors & more likely to overexpress

HER2/neu

• most carcinomas induce a marked increase in dense, fibrous desmoplasticstroma, giving the tumor a hard consistency on palpation and replace fat,resulting in a mammographic density (scirrhous carcinoma)


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Invasive Lobular Carcinoma

• present as a palpable mass or mammographic density

• produce only a vaguely thickened area of the breast or subtle architecturalchanges on mammography

• metastases can also be difficult to detect clinically and radiologically owing tothis type of invasion

• have a greater incidence of bilaterality

• increasing among postmenopausal women - may be related to the use ofpostmenopausal hormone replacement therapy

• Well-differentiated and moderately differentiated carcinomaso diploid, express hormone receptors, and are associated with LCISo HER2/neu overexpression is very rare

• Poorly differentiated lobular carcinomaso 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 andβ-catenin

• Metastases to the peritoneum and retroperitoneum, the leptomeninges(carcinomatous meningitis), the gastrointestinal tract, and the ovaries anduterus 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 onecell in width (Indian file)

• desmoplastic response may beminimal or absent

• cells have the same cytologicfeatures as LCIS and lack cohesion,without formation of tubules orpapillae

• signet-ring cells are common

• cells are arranged in concentric ringssurrounding normal ducts


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Metastasis

• lymphnode

• lungs

• bones

• liver

• adrenals• brain

• meninges

Prognostic FactorsMajor prognostic factors

o insitu / invasiveo distant metastasiso lymphnode metastasiso tumour sizeo local invasion

1. Invasive carcinoma or in situ disease

• in situ carcinoma cannot metastasize – associated with better prognosis.

• invasive carcinomas – associated with poor prognosis2. Distant metastases - associated with poor prognosis3. 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 disease6. Inflammatory carcinoma

• poor prognosis

Minor Prognostic Factorso histological subtypeo tumour gradeo ER/PR receptoro HER2/neu o lymphovascular invasiono proliferative rateo DNA content

• minor prognostic factors can be used to decide among chemotherapyregimens and/or hormonal therapies


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• Three of these factors—estrogen receptor, progesterone receptor, andHER2/neu—are most useful as predictive factors for response to specifictherapeutic agents.

Histologic subtypesSpecial types of invasive carcinomas (tubular, mucinous, medullary, lobular, and

papillary) has better prognosis than cancers of no special typeTumor gradewell-differentiated grade I tumors better than moderately differentiated grade IItumors, and poorly differentiated grade III tumorsEstrogen and progesterone receptorshormone receptor-positive cancers have a slightly better prognosisHER2/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/neudeveloped to specifically target tumour cells.

Lymphovascular invasion (LVI)strongly associated with the presence of lymph node metastases and is a poorprognostic factorProliferative ratehigh proliferation rates have a worse prognosisDNA contentaneuploid tumours have a worse prognosis

Treatment

• Mastectomy

• Radiation

•

Chemotherapy• Hormone therapy

• New strategies - (by pharmacologic agents or specific antibodies) ofmembrane-bound growth factor receptors (e.g., HER2/neu), stromalproteases, and angiogenesis


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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 metastaticpotential

• 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


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STROMAL TUMORS There are two types of stroma in the breast, intralobular and interlobular.

• Biphasic tumors arise in the interlobular stromao fibroadenomao phyllodes tumor

FibroadenomaIntro:

• most common benign tumor arise in the interlobular stromaClinical 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) developfibroadenomas

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 nodulesand contain slit like spaces

Micro:• stroma is cellular, and often

myxoid, resembling intralobularstroma

• glandular and cystic spaces lined

by epithelium

• epithelium may be surrounded bystroma or compressed anddistorted by it

• border is sharply delimited fromthe surrounding tissue


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Phyllodes Tumour (Syn. cystosarcoma phyllodes)

Introduction:

• term "phyllodes tumor" is preferred, as the majority of these tumors behavein a relatively benign fashion, and most are not cystic.

Origin: • tumour arises from intralobular stroma

• 6th decadeClinical Features:

• palpable massesMacroscopy: • 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 stromalovergrowth, 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 haematogenouso only the stromal component metastasizes

• axillary lymph node dissection is not indicated


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CastsIntroduction: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 bytubular 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 alsoincorporated.

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 severetubular damage

• WBC casts - acute pyelonephritis

• Granular and waxy casts - derive from renal tubular cell casts. When cellular castsremain 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 inend-stage chronic renal disease

Telescoped urinary sediment

• is one in which red cells, white cells, oval fat bodies, and all types of casts arefound in more or less equal profusion

• Occurs in 1) lupus nephritis 2) malignant hypertension 3) diabeticglomerulosclerosis, and 4) rapidly progressive glomerulonephritis


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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 cytologyIt deals with cells exfoliated from the surface. They are obtained by scraping the surface

or aspirating the fluid

Papsmear – cells exfoliated from cervixSputum – cells exfoliated from bronchopulmonary tree

Gastric lavage – Stomach

Pleural, Ascitic, CSF2. FNAC:

Cells are obtained by aspirating the lesion using a fine needle (23G) and a 10 ml syringee.g.,

• palpable lesions - breast, thyroid, lymphnode etc.• deep - lung, pancreas, liver, prostate, kidney etc.

Procedure:

Cells are made into smears on the slidesSmears 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 painfulLimitations:

• less sensitive than histopathology

• tissue architecture is not preserved

• false negative occurs, if the needle did not hit the target.


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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 gotdecreased.

• Platelets are also utilized in the coagulation and so reduced in number.

• Coagulation forms fibrin mesh inside the capillaries and the RBCs has to squeezethro’ it and get destroyed (haemolysed).

• At the same time, wide spread fibrinolysis also will take place. That leads toformation 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.


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Erythrocyte indices(Absolute values or Wintrobe’s constant)

MCV (Mean corpuscular volume)

• means volume of individual RBC

MCV = PCV in Litre / LitreRBC 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 = HbX10RBC 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/LOr

MCHC = Hb X 100PCV %

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


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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 – spherocytosisMethods

• Westergren’s method• Wintrobe method• MicroESR method• Automated method


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Frozen Section

Principle:

• when tissue is frozen, the water within the tissue turns to ice and in this state thetissue 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: (-15°C to -20°C)

1. Cryostat2. Freezing microtome

Technique:

• Preparation of tissue – either unfixed or fixed tissue. Rapid freezing by liquidnitrogen 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.


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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

patient’s 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• 37°C

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.


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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 leukaemiaand their number vary depends upon the type.

• Neutrophil alkaline phosphatase (NAP) activity – Increased in leukaemoidreaction whereas decreased in leukaemia.


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Leukocyte Cytochemistry

Introduction:

• Leukocyte cytochemistry encompasses the techniques used to identify diagnosticallyuseful 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 myelodysplasticsyndromes and myeloproliferative disorders.

• The use of cytochemistry to characterise lymphoproliferative disorders has beenlargely superseded by immunological techniques

• The results of cytochemical tests should always be interpreted in relation toRomanowsky 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 (α-Naphthyl Acetate Esterase) and ANBE (α-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. Pearl’s reaction - demonstration of ring sideroblasts in MDS (Blue)

7. Tartrate-Resistant Acid Phosphatase (TRAP) – positive in hairy cell leukaemia

(Brown)


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Packed Cell Volume (PCV) / HaematocritEquipments

• Wintrobe’s tube & Pasteur pipetteProcedure

1. Using the Pasteur pipette, fill the Wintrobe’s tube to ‘0’ mark

2. Centrifuge at 2000 to 2300g for 30 minutes3. 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 RBCcolumn is called buffy coat.

Uses of PCV

• to diagnose anaemia or polycythaemiaOther 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


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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 37°C 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 HbAppropriate 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.


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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 sectionsand 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


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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 aresponse to the presence of a tumour.

ClassificationTumour markers can be classified in two groups: Cancer-specific markers and tissue-

specific markers.

Cancer-specific markersCancer-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 orresponse 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 patientwith a history of a treated bowel cancer, a rising CEA level can be an early sign ofrecurring 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 nocancer 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 prostateo elevated beta-HCG – choriocarcinoma, hydatidiform mole, pregnancyo 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 treatmente.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.


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Limitations:

An elevated level of a tumour marker can indicate cancer; however, there can also beother 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 fluids2. Immunohistochemitry method – to detect the tumour markers in the tissue.

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