Endoscopic biopsy and git

2. Biopsy to microscopy
• Basic histology tour

6. ENDOSCOPY AND HISTOLOGY
• DIRECT EXAMINATION OF ORGANS WITH OPTICAL METHODS.
• DETECT ABNORMALITIES OF NORMAL ANATOMY AND HISTOLOGY TO PROVIDE A PRECISE DIAGNOSIS.
• CLASSICAL ENDOSCOPY--- NAKED EYE OBSERVATION
• HISTOLOGY IS REACHES CELLULAR AND SUBCELLULAR LEVEL
• MAJOR CONTRIBUTION OF HISTOLOGY TO ENDOSCOPY IN INFLAMMATORY AND NEOPLASTIC DISEASES.
• TYPE& ETIOLOGY OF INFLAMMATION AND CLASSIFICATION ,IMPACT ON TREATMENT,
• FOR eg: GLUTEN SENSITIVE ENTEROPATHY,IDENTIFYING SPECIFIC PATHOGENS LIKE GIARDIA,MYCOBACTRIUM,CRYTOPSPORIDIA,AMOEBAE
• DETERMINING ETIOLOGY OF INFLAMMATION AS AUTOIMMUNE GASTRITIS, OR TYPING TUMORS(ADENOCA OR
LYMPHOMA)
• ELEMENTS FOR FURTHER TREATMENT STRATEGY BY PRESENCE OR ABSENCE OF RISK FACTORS
• LIKE RESIDUAL TUMOR IN POLYPECTOMY SPECIMEN OR ENDOSCOPIC MUCOSAL RESECTION,DEMONSTRATIONS OF
MUTATIONS LIKE KRAS IN CRC OR HER2 AMPLIFICATION IN ESOPHAGEAL AND GASTRIC CA,USE OF BIOMARKERS.
• THESE APPLICATIONS HAVE IMPORTANT THERAPEUTIC IMPLICATIONS
• KRAS GENE ASSOCIATED WITH POOR RESPONSE,HIGH HER2 EXPRESSION BENEFITTED BY TRASTUZUMAB

7. INFLUENCE OF ENDOSCOPY ON DIAGNOSTIC
YEILD OF HISTOLOGY
• GENERAL REQUIREMENTS
• CLOSE COLLABORATION BETWEEN ENDOSCOPIST AND PATHOLOGIST
• COPY OF ENDOSCOPY REPORT MENTIONING ;
• SITE OF BIOPSY, MACROSCOPIC DESCREPTION OF LESION AND ADJACENT MUCOSA , AGE, IMMUNE STATUS,
DURATION OF SYMPTOMS AND TREATMENT IF ANY.
• PATHOLOGIST SHOULD MENTION NUMBER,SIZE AND DEPTH OF SPECIMEN,
• PROBABILITY OF INITIAL DIAGNOSIS,SUGGEST FURTHER NEED FOR SAMPLING,IHC.

8. RECOMMENDATION FOR BIOPSIES STRATEGIES IN INFLAMMATORY CONDITION OF
THE GIT.

9. ENDOSCOPIC ULTRASOUND GUIDED FNAB.
• MOST ACCURATE MODALITY FOR CHARACTERIZATION OF PANCREATIC CYSTIC AND SOLID LESIONS, D/D
INTERMINATE MASSES AND LOCO-REGIONAL STAGGING OF SOME DIGESTIVE CANCERS.
• CAN BE PERFORMED IN THE PRIMARY MASS, DISTANT LYMPHNODES OR METASTATIC LOCATION.
• HIGH SENSITIVITY, SPECIFICITY, POSITIVE PREDECTIVE VALUE AND ACCURACY IN THE ASSESMENT OF
BILIOPANCREATIC TUMOR (DEPEND UPON THE EXPIRIENCE) OF CLINICIAN AND CYTOPATHOLOGIST.
• NUMBER OF SAMPLES:- DIAGNOSTIC YEILD OF HISTOPATHOLOGY IS INCREASED AND SAMPLING ERROR IS
DECREASED BY INCREASING THE NUMBER OF BIOPSIES.
• DIFFERENT GUIDELINES FOR ENDOSCOPIC SAMPLING IN VARIOUS DISEASES HAVE BEEN DEVELOPED.
• INTRODUCTION OF NEW TECHNOLOGY AND MODERN ENDOSCOPE WILL CHANGE PRACTICE IN FUTURE BY
OFFERING TARGETED BIOPSIES.

10. HOW TO SUBMIT SPECIMENS FOR HISTOPATHOLOGY.
• REQUISITION FORM / SUBMISSION FORM.
• CONTAINER.
• FIXATIVE.
• PROVIDE ANATOMICAL SITE, LESION DESCRIPTION AND PERTINENT CLINICAL INFORMATION ON THE
SUBMISSION FORM.
• IF YOU HAVE A LIST OF DIFFERENTIALS YOU’D LIKE TO RULE OUT, PLEASE MENTION SUCH.
• FIRST OF ALL AND MOST IMPORTANTLY CLINICIAN SHOULD TAKEN ADEQUATE CARE TO AVOID
CONTAMINATION OF TISSUE WITH TISSUE FROM OTHER PATEINT.
• THIS MAY HAPPEN IN OPERATION ROOM, CLINIC, OR IN PATHOLOGY LAB.

12. SAMPLING CONTINUED
• A COMPLETE DIAGNOSIS INVOLVES;
TUMOR DIFFERENTIATION, SIZE, DETERMINATION OF DEEP
INFILTRATION, LYMPHATIC PERMEATION AND DETERMINATION OF
MARGIN (IDENTIFICATION OF THIS AREA IS EASY IF LESION IS
ADEQUATELY ORIENTED).
• POLYPECTOMY- ENDOSCOPIST SHOULD IDENTIFY
SECTION MARGINS WITH INDIA INK / PIN.
• EMR / ESD SPECIMEN SHOULD BE ORIENTED
PROPERLY
• PAINTING OF BASE AND MARGIN IS USEFUL.

13. SPECIMEN CONTAINER.
• PLASTIC OR GLASS JAR.
• LABEL MATCHING REQUISITION SLIP.
• REGISTERATION NUMBER.
• FULL NAME.
• AGE / SEX
• WARD NUMBER, & BED NUMBER.
• SITE AND SIDE.
• MORE SPECIMEN MARK AS A,B,C,D ETC.
• SIGNATION OF DOCTOR WITH DATE.

14. SAMPLING
• DIAGNOSTIC YEILD DEPENDS UPON EXPERIENCE ,QUALITY OF BIOPSY.
• PROBABILITY OF INITIAL DIAGNOSIS, TYPE OF BIOPSY, SIZE OF BIOPSY FORCEPS.
• ANATOMIC LOCATION OF CERTAIN LESION ARE OF LESS OF GOOD QUALITY OR
SUPERFICIAL FOR EXAMPLE AREAS IMMEDIATELY DISTAL TO A STRICTURE, PAPILLA OF
VATER, PANCREATIC DUCTS ETC.
• TO OBTAIN SAMPLES OF APROPIATE DEPTH, AIR IN INSUFFLATION DURING THE
ENDOSCOPY SHOULD BE THE LIMITED MUCOSA IS STRETCHED AND PUSHED TOWARDS
SUBMUCOSA AND THE SAMPLES ARE LIKELY TO BE THE SUPERFICIAL.
• USE OF ‘BURROWING’ TECHNIQUE- SEVERAL BIOPSY ARE TAKEN IN THE SAME AREA GIVING
INFLAMMATION DEEPLY SITUATED LESION.
• EUG FNA IS AN ALTERNATIVE; PERMITING MORPHOLOGIC AND CYTOLOGIC ANALYSIS OF LESIONS
WITHIN OR ADJACENT TO GIT.
• LARGER SAMPLES OBTAINED WITH EMR / ESD / SNARE POLYPECTOMY.
• PROPER HANDLING AND INTERPRETATION OF THIS SAMPLES HELP SUBSEQUENT MANAGEMENT.

15. SPECIMEN IDENTIFICATION AND LABELLING
• ANY DISCREPANCIES OF SPECIEN IDENTIFICATION NOTED BY PATHOLOGY ASSISTANT SHOULD CONTACT WITH
PATHOLOGISTS AND / OR CLINICIAN OF THERE ARE ANY QUESTIONS.
CAUSES OF REJECTION OF SPECIMEN
• SPECIMEN NOT IN FORMALIN.
• UNLABELED OR IMPROPERLY LABELED CONTAINER.
• WITHOUT REQUISITION SLIP OR INCOMPLETE REQUISITION SLIP.

16. ENDOSCOPIC BIOPSIES.
• ALL FRAGMENTS SHOULD BE SUBMITTED IN
THE SAME CONTAINER.
• NUMBER OF FRAGMENTS, AGGREGATE
DIMENSION.
• COLOUR AND CONSISTENCY SHOULD NOT BE
CUT OR INKED.
• ALL SMALL BIOPSIES MUST BE SUPPORTED
WITHIN THE CASSETTES TO PREVENT TISSUE
LOSS DURING PROCESSING.
• SMALL FRAGMENTS MAY BE DIPPED IN EOSIN
TO MAKE THEM VISIBLE.
• DO NOT SUBMIT ENDOSCOPIC BIOPSIES
WRAPPED IN GAUZE.

18. ORIENTATION OF SPECIMEN

20. • TO BE ABLE TO STUDY MICROSCOPIC FEATURES OF TISSUE IT SHOULD
UNDERGO CERTAIN STEPS.
• WE ARE GOING TO TALK ABOUT THESE STEPS BRIEFLY.

23. TISSUE FIXATION.
• SHOULD PREVENT AUTOLYSIS AND PUTREFACTION OF THE CELL.
• SHOULD PENETRATE EVENLY AND RAPIDLY.
• SHOULD HARDEN THE TISSUES.
• INCREASE THE OPTICAL DENSITY.
• SHOULD NOT CAUSE SHRINKAGE OR SWELLING OF THE CELLS.
• MUST NOT REACT WITH THE RECEPTOR SITES AND THIS MUST NOT INTERFERE
WITH THE STAINING PROCEDURE.
• MUST BE CHEAP AND EASILY AVAILABLE.

26. TISSUE FIXATION.
• SPECIMEN SUBMIT IN 10% FORMALIN.
• FORMALIN TISSUE RATIO 10:1
• NO OTHER FIXATIVE SHOULD BE USED.
• SPECIMEN SHOULD BE IN A CONTAINER THAT CAN BE SEALED AND WILL NOT LEAK.
FRESH TISSUE TO BE SUBMITTED IN THE CASES OF:-
• FROZEN SECTION
• CULTURES
• IMMUNOFLOURESCENCE
• FLOWCYTOMETRY
• CHROMOSOME STUDIES
• ELECTRON MICROSCOPY

27. What is tissue processing?
• Tissue processing is a procedure of removing water from cells and
replacing it with a medium which solidifies allowing thin sections to
be cut on a microtome.
• Tissue processing is routinely done on an instrument called Tissue
Processor.

36. TISSUE PROCESSING
• THE TECHNIQUE OF GETTING FIXED TISSUE INTO PARAFFIN IS CALLED TISSUE PROCESSING, THE MAIN STEPS
IN THIS PROCESS ARE DEHYDRATION AND CLEARING.
• WET FIXED TISSUES CANNOT BE DIRECTLY INFILTRATED WITH PARAFFIN, FIRST THE WATER FROM THE
TISSUES MUST BE REMOVED BY DEHYDRATION.
• THE NEXT STEP IS CALLED “CLEARING” AND CONSISTS OF REMOVAL OF THE DEHYDRANT WITH A SUBSTANCE
THAT WILL BE MISCIBLE WITH THE EMBEDDING MEDIUM (PARAFFIN).
• FINALLY THE TISSUE IS INFILTRATED WITH THE EMBEDDING AGENT, ALMOST ALWAYS PARAFFIN.

37. • TISSUES THAT COME OFF THE TISSUE PROCESSOR ARE STILL IN THE CASSETTES AND MUST BE MANUALLY
PUT INTO THE BLOCKS BY A TECHNICIAN WHO MUST PICK THE TISSUES OUT OF THE CASSETE AND POUR
MOLTEN PARAFFIN OVER THEM.
• THIS “EMBEDDING” PROCESS IS VERY IMPORTANT, BECAUSE THE TISSUES MUST BE ALIGNED, OR ORIENTED,
PROPERLY IN THE BLOCK OF PARAFFIN.

39. SECTIONING
• ONCE THE TISSUES HAVE BEEN EMBEDDED, THEY MUST BE
CUT INTO SECTIONS THAT CAN BE PLACED ON A SLIDE.
• THIS IS DONE WITH A MICROTOME, THE IMPORTANT THING
FOR PROPER SECTIONING IS A VERY SHARP KNIFE.
• MICROTOMES HAVE A MECHANISM FOR ADVANCING THE
BLOCK ACROSS THE KNIFE, USUALLY THIS DISTANCE CAN BE
SET, FOR MOST PARAFFIN EMBEDDED TISSUES AT 6 TO 8
MICRONS.
• SECTIONING TISSUES IS A REAL ART AND TAKES MUCH SKILL
AND PRACTICE, IT IS IMPORTANT TO HAVE A PROPERLY FIXED
AND EMBEDDED BLOCK OR MUCH ARTEFACT CAN BE
INTRODUCED IN THE SECTIONING.
• COMMON ARTEFACTS INCLUDE TEARING, HOLES, FOLDING
ETC.
• ONCE SECTIONS ARE CUT THEY ARE FLOATED ON A WARM
WATER BATH THAT HELPS REMOVE WRINKLES.
• THEN THEY ARE PICKED UP ON A GLASS MICROSCOPIC SLIDE.
• THE GLASS SLIDES ARE THEN PLACED IN A WARM OVEN FOR
ABOUT 15 MINUTES TO HELP THE SECTION ADHERE TO THE
SLIDE.

41. Staining
If the slide is examined under microscope without
staining we can see only vague shadows of the tissue
To visualize the tissue and its cellular components we
have to use dyes which stain the different components
according to their chemical compositions

42. STAINING AND COVER SLIPPING
• THE EMBEDDING PROCESS MUST BE REVERSED IN ORDER TO GET THE PARAFFIN WAX OUT OF THE TISSUE
AND ALLOW WATER SOLUBLE DYES TO PENETRATE THE SECTIONS.
• THEREFORE, BEFORE ANY STAINING CAN BE DONE, THE SLIDES ARE “DEPARAFFINIZED” BY RUNNING THEM
THROUGH XYLENES TO ALCOHOLS TO WATER.
• THERE ARE NO STAINS THAT CAN BE DONE ON TISSUES CONTAINING PARAFFIN.
• THE STAINED SECTION ON THE SLIDE MUST BE COVERED WITH A THIN PIECE GLASS TO PROTECT THE TISSUE
FROM BEING SCRATCHED, TO PROVIDE BETTER OPTICAL FOR VIEWING UNDER THE MICROSCOPE AND TO
PRESERVE THE TISSUE SECTION FOR YEARS TO COME.
• THE STAINED SLIDE MUST GO THROUGH THE REVERSE PROCESS THAT IN WENT THROUGH FROM PARAFFIN
SECTION TO WATER.

49. FROZEN SECTION
• FROZEN SECTIONS ARE PERFORMED WITH AN
INSTRUMENT CALLED A CRYOSTAT.
• THE CRYOSTATE IS JUST A REFRIGERATED BOX
CONTAINING A MICROTOME.
• THE TEMPRATURE INSIDE THE CRYOSTATE IS
ABOUT -20 TO -30 DEGREE C.
• THE TISSUE SECTIONS ARE CUT AND PICKED UP ON
A GLASS SLIDE.
• THE SECTIONS ARE DRIED AND THEN STAINED.

50. MICROWAVE PROCESSING
• WITHIN THE LAST DECADE, TISSUE PROCESSING WITH THE MICROWAVE OVEN HAS BEEN INTRODUCED INTO
HISTOLOGY LABORATORIES.
• PROCESSING TIMES ARE SIGNIFICANTLY REDUCED BUT THROUGHPUT IS VERY LOW.
• ONLY LABORATORY MICROWAVE OVERNS SHOULD BE USED AS THE TEMPRATURE MUST BE CAREFULLY
CONTROLLED AND THE MICROWAVE MUST BE VENTED JUST LIKE A CHEMICAL FUME HOOD.
• THIS TECHNOLOGY IS ESPECIALLY USEFUL FOR BIOPSY SIZED SPECIMENS.
• REAGENTS USED IN MICROWAVE PROCESSING INCLUDE:- ETHYL ALCOHOL, ISOPROPYL ALCOHOL AND
PARAFFIN.

51. • Proper orientation of the tissue samples is important for a
correct diagnosis of malabsorptive states such as celiac
disease, where the ratio villous height – crypt depth must be
assessed and for specimens from endoscopic resections of
polyps or early neoplastic lesions.
• Multiple sections from multiple endoscopic biopsies allow a
more complete microscopic analysis

52. Immunohistochemistry and other ancillary
techniques
• Histopathology is an adequate tool for solving differential diagnostic
problems and typing of tumours.
• Anaplastic carcinomas, large-cell lymphoma, epithelioid stromal tumours
and neuroendocrine tumours can be difficult but immunohistochemical
stainings with antibodies against cytokeratins (CK), a marker for epithelial
cells,
• CD117 a marker for gastrointestinal stromal tumors,
• chromogranin, a marker for endocrine cells and a
• common leucocyte marker can solve the problem.
• Antibodies to intermediate filaments such as the CKs can be potentially
useful in other situations. CKs comprise a subfamily of more than 20
members.
• CK7 and CK20 & examination of coordinate expression of these two CKs
can help in the differential diagnosis of carcinomas of unknown primary
site.

53. • Immune histochemistry and cytogenetic analysis is essential for the
management of lymphomas. Primary intestinal lymphomas should
be sub-typed in B cell and T cell malignancies and classified
according internationally validated classifications such as the
recently published WHO.
• Evaluating the proliferation fraction of the tumour cells using a
marker such as Ki67 or MIB1 may provide some additional
information on the biological behaviour of lymphomas. This is also
true for endocrine tumours and gastrointestinal stromal tumours.
Further ancillary techniques may include staining with antibodies
against p53 for Barret’s oesophagus or colitis-associated dysplasia.
Currently a number of markers are under investigation for a more
accurate identification of early neoplasia.

54. • Histochemistry (histological special stains) searching for mucins or
other substances, and occasionally electron microscopy and genetic
markers can also be applied on biopsy samples. Many stainings can
be performed on routinely formalin fixed material. Increasingly there
is some overlap, between immune histochemistry and molecular
techniques since genetic markers can be demonstrated also by
immune histochemistry. This is for instance true for large-bowel
cancers with microsatellite instability (MS), where the products of the
DNA repair genes hMLH1, hMSH2 and MSH6, or the lack of them,
can be demonstrated immune histochemically. These products do
not however cover the whole range of MS. DNA or RNA extraction
and genetic analysis remains important and there may even be a
growing need.