8/19/2019 Misc Answers
1/33
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e) The stylet is withdrawn and a 10ml syringe is attached to the needle.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 Then suction is stopped.f) Needle and syringe together are withdrawn and marrow is poured onto the slides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
8/19/2019 Misc Answers
2/33
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
8/19/2019 Misc Answers
3/33
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
8/19/2019 Misc Answers
4/33
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 (
8/19/2019 Misc Answers
5/33
8/19/2019 Misc Answers
6/33
• 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
8/19/2019 Misc Answers
7/33
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%
8/19/2019 Misc Answers
8/33
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
8/19/2019 Misc Answers
9/33
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
8/19/2019 Misc Answers
10/33
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
8/19/2019 Misc Answers
11/33
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
8/19/2019 Misc Answers
12/33
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)
8/19/2019 Misc Answers
13/33
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
8/19/2019 Misc Answers
14/33
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
8/19/2019 Misc Answers
15/33
• 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
8/19/2019 Misc Answers
16/33
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
8/19/2019 Misc Answers
17/33
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
8/19/2019 Misc Answers
18/33
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
8/19/2019 Misc Answers
19/33
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
8/19/2019 Misc Answers
20/33
8/19/2019 Misc Answers
21/33
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.
8/19/2019 Misc Answers
22/33
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.
8/19/2019 Misc Answers
23/33
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
8/19/2019 Misc Answers
24/33
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
8/19/2019 Misc Answers
25/33
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.
8/19/2019 Misc Answers
26/33
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.
8/19/2019 Misc Answers
27/33
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.
8/19/2019 Misc Answers
28/33
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)
8/19/2019 Misc Answers
29/33
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
8/19/2019 Misc Answers
30/33
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.
8/19/2019 Misc Answers
31/33
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
8/19/2019 Misc Answers
32/33
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.
8/19/2019 Misc Answers
33/33
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.