TISSUE AND MOLECULAR

DIAGNOSIS
• TISSUE DIAGNOSIS, IMPROVED FROM MACROSCOPIC EXAMINATION OF AUTOPSY
MATERIAL TO MICROSCOPIC EXAMINATION OF AUTOPSY AND SURGICAL
PROCEDURE
• MODERN CELLULAR PATHOLOGY DEPARTMENT LOCATED IN HOSPITAL
COLLECTS SPECIMENS
 REASON FOR TISSUE ANALYSIS

• TO MAKE NEW DIAGNOSIS

• CONFIRM SUSPECTED / ESTABLISHED DIAGNOSIS
• EXCLUDE ADDITIONAL DIAGNOSES
• ASSIST WITH PROGNOSIS
• TO STAGE TUMORS
• SELECT THERAPY AND MANAGEMENT
• ASSESS RESPONSE TO TREATMENT
 REQUEST FORM WITH CLINICAL DETAILS
SHOULD ACCOMPANY ALL SPECIMENS
• SITE OF BIOPSY
• DATE
• GENDER
• MEDICATIONS
• PAST MEDICAL AND SURGICAL HISTORY AND DETAILS OF NEO ADJUVANT
THERAPY IN CVANCER CASES
 Tissue samples

Histopathology Cytopathology

Fresh tissue – fresh FNAC, Cervical , fluids/

Biopsy Resections - frozen sputum , washings , scrapes

Mucosal ,Punch,Needle
Currettings, excision biopsy
SPECIMEN PROCESSING
 HISTOLOGY SPECIMEN
Biopsy / resection
Stained with H and E Examines slides
received

Histology compares
Macroscopic Sections put on glass
with clinical and
description made slides
macroscopic findings

5 micr0metre sections Report entered ,

Specimen sampled
cut authorized / signed

Paraffin wax blocks

Placed in cassette
made
 FROZEN SECTION

• ADVANTAGES
• QUICK DIAGNOSIS
• DISADVANTAGE- HIGH RISK OF INFECTION
• POORER QUALITY SECTIONS
• SMALL SAMPLE REQUIRED
• SOME TISSUES DIFFICULT TO PROCESS
 CYTOLOGY SPECIMEN

• CYTOLOGY SPECIMEN FIXED BY ALCOHOL OR AIR DRIED

• STAINED WITH PAP STAIN OR MAY GRUNWALD GIEMSA OR ROMANOWSKY STAIN
• MOVED TO LIQUID BASED THIN LAYER TECHNOLOGY
• LIQUID BASED SAMPLES WASHED IN A LIQUID MEDIUM AND MATERIAL
OBTAINED GETS PROCESSED
 STORAGE

• RESECTION SPECIMENS STORED FOR 4- 6 WEEKS

• TISSUE BLOCKS STORED LONGER PERIODS – EVEN 30YEARS
• GLASS SLIDES – SHORTER PERIOD
• USE OF STORED SPECIMENS – FUTURE CLINICAL REVIEW
• TEACHING
• AUDIT , RESEARCH PURPOSES
 PRINCIPLES OF MICROSCOPIC DIAGNOSIS

• DIAGNOSIS OF MALIGNANCY
• NON NEOPLASTIC AND INFLAMMATORY CONDITIONS
 MICROSCOPIC FEATURES OF MALIGNANCY

• METASTASIS T ANOTHER ORGAN/ LYMPH NODES

• INVASION OF SURROUNDING STRUCTURES , VASCULAR , PERINEURAL INVASION
• ARCHITECTURAL ABNORMALITIES
• NECROSIS
• NUMEROUS MITOTIC FIGURES
• ATYPICAL MITOTIC FIGURES
• CELLULAR LEVEL CHANGES – INCREASED NUCLEAR CYTOPLASMIC RATIO ,NUCLEAR
ABNORMALITIES
 CAUSES OF FALSE POSITIVE DIAGNOSIS OF
MALIGNANCY
• INTERCHANGEED SAMPLES
• CONTAMINATION
• INTERPRETOR ERROR
• TREATMENT INDUCED CHANGE
• ULCERATION
• HISTOLOGY TYPES OF MALIGNANCY
• PROGNOSTIC FACTORS FOR MALIGNANT FEATURES
MICROSCOPIC FEATURES OF INFLAMMATIONS

• ACUTE INFLAMMATION – NEUTROPHILS

• CHRONIC INFLAMMATION – EOSINOPHILS , MAST CELLS , HISTIOCYTES
• GRANULOMAS – COLLECTION OF EPITHELOID HISTIOCYTES
 TISSUE ASSESSMENT

Tissue assessment

Cytological assessment
Light microscopy Histological assessment Architecture not seen
-architecture assessed Archtecture assessed but acharacters can be
assessed
 ADVANTAGE AND DISADVANTAGES OF
CYTOLOGY OVER HISTOLOGY
• ADVANTAGES
• WIDER AREAS SAMPLED
• SAMPLING LESS INVASIVE
• FAST
• CHEAP
• DISADVANTAGES- ARCHITECTURE CANNOT ASSESSED , AND LESS AMENABLE
FOR FURTHER STUDIES
• TISSUE DIAGNOSIS ALSO USED FOR SCREENING PURPOSES
• TO DETECT AND TREAT PRE MALIGNANT TISSUE CHANGES
 ADDITIONAL TECHNIQUES

• SPECIAL STAINS
• IMMUNOHISTOCHEMISTRY –
• ELECTRON MICROSCOPY
• MOLECULAR PATHOLOGY
• FISH – IN SITU HYBRIDIZATION
• PCR BASED THECHNIQUES
 STAINING TECHNIQUES

• PAS – GLYCOGEN , FUNGI

• ‘D-PAS – MUCIN
• PRUSSIAN LUE – IRON
• RETICULIN – RETICULIN FIBRES
• VAN GIESON – COLLAGEN
• CONGO RED – AMYLOID
• ZIEHL NELSON – MYCOBACTERIA
 ELECTRON MICROSCOPY

• HIGH MAGNIFICATION
• DIFFERENTIATE NEOPLASTIC AND NON NEOPLASTIC
• DISADVANTAGE –
• TIME CONSUMING
• LABOUR INTENSIVE
• EXPENSIVE
 IMMUNO HISTOCHEMISTRY

• DETECTS SPECIFIC ANTIGEN USING AN ANTIBODY

• ANTIBODY LABELLED WITH DYE , WEN BOUND TO ITS TARGET ANTIGEN SEEN IN
TISSUE SECTION AS COLOURED STAIN
• TISSUE DISTRIBUTION , CELLULAR LOCALIZATION OF AN ANTIGEN DETERMINED
 USES OF IMMUNOHISTOCHEMISTRY

• CELL TYPE
• NEOPLASIA – DIFFERENTIATION
• SITE OF ORIGIN DETERMINATION
• NEOPLASIA CONFIRMATION
• TREATMENT SELECTION
• SCREENING FOR MUTATION
• PROGNOSIS
• MICROORGANISMS DETECTIONS EG- CMV , EBV
 MOLECULAR PATHOLOGY

• MULTIPLE TESTS ASSESS MOLECULES – PROTEINS , RIBONUCLEIC ACID , DNA IN

TISSUE
• USEFUL FOR DIAGNOSIS
• PROGNOSTIC PREDICTIONS
• HEREDITARY CANCER RISKS
• DETERMINING TREATMENT
• RESIDUAL DISEASE AFTER TREATMENT
 METHODS

• IN SITU HYBRIDIZATION
• LABELLED OLIGONUCLEOTIDE PROBE TARGETED AT A SPECIFIC SEQUENCE OF
RNA OR DNA
• AUTORADIOGRAPHY , FLUORESCENCE MICROSCOPY USED FOR VISUALIZATION
 POLYMERASE CHAIN REACTION

• AMPLIFIES DNA , YIELDS MILLION OF COPIES FROM SINGLE COPY OF SELECTED

TARGET
• RNA ALSO AMPLIFIED , USING THE TECHNIQUE OF REVERSE TRANSCRIPTASE PCR
• FAST AND SAFE IN HOMOGENISED FRESH OR FORMALIN FIXED TISSUE
• DETECTS TRANSCRIPTION
• AMPLIFICATIONS
• DEMONSTRATION OF MICROSATELLITE INSTABILITY
• DETECTION OF GENE HYPERMETHYLATION
• DETECT MICRO ORGANISMS
 CYTOGENETICS AND FISH

• CYTOGENETICS IS THE MICROSCOPIC STUDY OF CHROMOSOMAL CHANGES IN

INDIVIDUAL CELLS
• DETECTS GENE AMPLIFICATIONS
• LOSS OF SEGMENTS OF CHROMOSOMAL MATERIAL
• LOSS OF WHOLE CHROMOSOMES AND TRANSLOCATIONS AND FUSION GENES
DETECTED
 FLOW CYTOMETRY

• LASER OR IMPEDANCE BASED TECHNIQUE USED FOR IN ENGINEERING

• DETERMINES PLOIDY AND ANTIGENS IN HAEMATOLOGICAL NEOPLASMS
• CELLS ARE SUSPENDED IN A STREAM OF FLUID AND PASSED BY AN ELECTRONIC
DETECTION APPARATUS
 EXAMPLES

• LYMPHOMA – IMMUNOGLOBIN H CHAIN REARRANGEMENTS IN B CELL

PROLIFERATION
• IN SOFT TISSUE SARCOMAS
• GISTS – KIT GENE MUTATION
• PREDICTION OF RESPONSE TO DRUG THERAPY FOR ADVANCED CARCINOMAS
THANK YOU