Histopathology specimen processing

Histopathology specimen processing
AZFAR NEYAZ, JUNIOR RESIDENT
SGPGIMS, LUCKNOW

 Specimen identification and labeling
 Grossing & Fixation
 Dehydration
 Clearing
 Impregnation
 Embedding
 Microtomy
 Staining and Mounting
Outline

Specimen identification and labeling
 Tissue specimen received in the laboratory have a request form that
lists the patient information, history & description of the site of origin.
The specimen are accessioned by giving
them a number that will identify each
specimen for each patient

Information must be provided
 Patient Identification
 Identification of individual requesting examination
 Procedure date
 Adequate clinical history and physical examination
 Organs resected or biopsied
 Orientation if required.
 Intraoperative findings
 Prior surgery or pathologic diagnosis
 Prior treatment
 Mention if rapid diagnosis is required.

• Complex series of chemical events to preserve the basic structure of
the tissue in such a manner that subsequent examination may be
made to determine the microanatomy and allow localisation of its
various chemical constituents
• The purpose of fixation is to preserve tissues permanently in life-like
state as possible
Fixation of tissues

Ideal Fixatives
 Prevent autolysis & bacterial decomposition
 Preserve tissue in their natural state & fix all components
 Make cellular components insoluble in liquids encountered in tissue
processing
 Preserve tissue volume
 Avoid excessive hardness of fixed tissue
 Enhance staining of tissues
 Non-toxic & non-allergic

Types of fixatives
• Physical methods:
– Heating, Microwaving, Freeze drying
Chemical methods
• Coagulatant fixatives :(Precipitate and coagulate protein)
– Alcohols and acetone
– Picric acid and trichloro acetic acid
• Cross linking fixatives:
– Formaldehyde and glutaraldehyde
– Other aldehydes (chloral hydrate, glyoxal)
– Metal salts (Mercuric and zinc chloride, osmium tetroxide)
• Compount fixatives

• 10% neutral buffered form(NBF) is most commonly used fixative
• It is prepared by mixing 40% formaldehyde gas in 100 w/v of distilled
water.
• The resultant mixture is 100% formalin.
• Routinely, 10% formalin is used which is prepared by mixing 10ml of
100% formalin in 90ml of distilled water.
Mechanism of action:
• It forms cross links between amino acids of proteins thereby making
them insoluble.
• It fixes 4 mm thick tissue in 8 hours .
• The fixative should be 10 times more in volume then the specimen.
Formalin

Advantages:
• Rapid penetration
• Easy availability & cheap
• Does not overharden the tissue
• Fixes lipids for frozen sections
Disadvantages:
• Irritant to the nose, eyes and mucous membranes
• Formation of precipitate of paraformaldehyde (which can be
prevented by adding 11- 16% methanol)
• Formation of black formalin pigment (acid formaldehyde hematin).

Other Fixatives
• Glutaraldehyde and Osmium Tetraoxide
• B5 fixative:
– stock solution: Mercuric chloride, sodium acetate & distilled water
– Add 2ml of formaldehyde to 20ml of stock solution
• Zenker's fluid
• Bouin’s Fluid

Decalcification
• It is the process of removal of the calcium salts from the specimen.
• The various agents used for decalcifying are:
• Nitric acid
• Hydrochloric acid
• Formic acid
• Picric acid
• Acetic acid
• Citric acid

Grossing
• Grossing of specimen is important stage in surgical pathology.
• It involves:
– Accurate naked eye description of intact specimen
– Correct method of sectioning
– Gross examination of cut surface
– Selection of proper tissue blocks for microscopy
– Instructions for embedding & block making.
• Histopathology specimens are a vital in patient care.
• They not only establish tissue diagnosis, but are crucial in clinical
management & provide important prognostic data.

Grossing is an art
A knowledge of what needs be taken for microscopic study is
crucial for final diagnosis.

 Large room, well illuminated and properly ventilated.
 Cutting board placed inside a metal box designed in such a fashion
that all the fluids flow directly into the sink.
 Shelves for specimen container.
 Ready access to a sink with hot and cold water.
 Ready access to formalin
 Box of instruments, Box with cassettes, labels.
 Large formalin container, photographic facilities.
 Large table with sink for large specimens
 Central table for multiple purpose.
Grossing room

Large scissors
Dissecting
scissors
Enterotome
Saw
scalpel
Instruments used in grossing

Forceps
Ruler
BLADE
Cutting
board
GLOVES
Instruments used in grossing

Inking
• Resection margins
• Embedding instructions
• Orientation
• Distinguish between samples
• Identify the cut surface

General principles of grossing
• Specimen identification
• Identify all the anatomical structures present
• Orientation markers should be identified
• Measurements: Length, breadth, height
• Weight-especially parenchymatous organ
• Examine the external surface.
• Cut all the organs at intervals of 1cm thickness
• Describe cut surface, identify pathologic process
• If suspected lesion is present, measure, describe with colour &
consistency.
• Surgical margins
• Histological sections

 Number of fragments, aggregate dimension
 Greatest dimension of largest fragment
 Shape of the fragment
 Colour and consistency
 Should not be cut or inked
 All small biopsies must be supported within the cassette to prevent
tissue loss during processing.
 All fragments are submitted.
 Small fragments may be dipped in eosin to make them more visible.
Small biopsies

Lymph node
1. If the lymph node is received in the fresh state, cut 2–3mm slices
perpendicular to the long axis-
• Take a small portion for culture
• Make imprints smears from the cut surface
• Submit tissue for flow cytometry, cytogenetics & molecular genetics
• Rest of tissue fix in formalin, and submit for histology.
2. If the specimen is received in formalin, submit representative sections.
Description
 State whether node received fresh or fixe
 Size, number of node and condition of capsule
 Appearance of cut surface: color, nodularity, hemorrhage, necrosis
Sections for histology
• One to three sections including capsule, depending on size of node

• Cut parallel slices along the long axis through hilum while the
specimen is fresh and examine each slice carefully for focal lesions
Description
• Weight and dimensions
• Hilum: Nature of vessels, presence of lymph nodes, presence of
accessory spleens
• Capsule: color, thickness, focal changes, adhesions, lacerations
• Cut surface: color, consistency, bulging, fibrous trabeculae, nodules
or masses, diffuse infiltration
Spleen – splenectomy

Incidental splenectomy: one section, including capsule
Traumatically ruptured spleen:1 section through tear & 1away from it
Diseased spleen: at least 3 sections, 1 from hilum & two from capsule
Suspected Lymphoma : All grossly abnormal/suspicious areas; if no
gross abnormalities seen, four random pieces

Thymus gland – thymectomy
Procedure
• Weigh the entire organ. Cut parallel slices
• Look carefully for lymph nodes around the thymus
Description
• Weight and dimensions; both lobes identifiable?
• Relative amount of fat and thymic parenchyma
• Tumour characteristics: size, shape, external appearance
• Cut section:color, necrosis,hemorrhage, fibrous band, calcification
• Attached structures (pleura, pericardium, lung, lymph nodes)

Sections for histology :
• Tumor: 3 or more sections, at
least two of which should include
capsule
• Uninvolved thymus: 2 sections
• Other organs, if present (lung,
lymph nodes)

<4 mm
4-8 mm
Skin – excision for benign lesion
Specimens upto 3mm
Specimens upto 3-6mm
Specimens more than 6mm

Skin – excision for malignant tumor
Small specimens – up to 5cm in greatest length

Thyroid gland–Thyroidectomy
Procedure
• Weigh and measure the specimen
• Orient the specimen and cut parallel longitudinal slices 5 mm each
• Search for parathyroid glands in the surrounding fat
Description
• Type of specimen: lobectomy, subtotal thyroidectomy, total
thyroidectomy
• Weight, shape, size, color and consistency of specimen
• Cut surface: smooth or nodular
• if nodular: number, size & appearance of nodules(cystic, calcified,
hemorrhagic, necrotic)
• Encapsulated or invasive, distance to line of resection

For diffuse/Inflammatory lesions
Three sections from each lobe and one from isthmus

For multinodular thyroid glands
• One section of each nodule (up to five nodules), including rim
& adjacent normal gland.
• More than one section for larger nodules

Solitary Encapsulated Lesion
• For a solitary encapsulated nodule measuring up to 5cm: Entire
circumference
• Most of these sections should include the tumor capsule and
adjacent thyroid tissue
• 1 additional section for each additional centimeter in diameter.

Grossly invasive carcinoma other than papillary
• Three sections of tumor
• Three of non-neoplastic gland
• One or more from line of resection

For papillary carcinoma
•The glands needs to be sampled extensively due to
multicentricity of tumour.
•Minimum 5 bits to be taken from gland proper in addition to those
from line of resection.

Parathyroid glands– Parathyroidectomy
• Removal of diseased parathyroid glands is usually complete, but in
cases of hyperplasia a portion of one gland is retained
Procedure
• Accurately weigh each gland on a delicate balance after removing the
surrounding fat
• Accurately label each parathyroid gland as to site
Description
• Weight, color, consistency, and external appearance of each gland
• All parathyroid tissue
• Except for markedly enlarged gland in which minimum of three
sections should be taken

Adrenal gland– adrenalectomy
Procedure :
• Ink the specimen
• Cut parallel sections at 5 mm intervals in the transverse plane
Description :
• Size and weight
• External surface: smooth, bosselated, shape of gland preserved
• Cut surface: color, necrosis, hemorrhage, cystic changes,
encapsulation, extension into the surrounding tissues
• Tumor if present, trying to demonstrate relationship with normal
gland, tumor capsule, and surrounding organs
• Non-neoplastic gland, some including adrenal vein

Esophagus – Esophagectomy
Procedure
• Dissect the specimen in the fresh state
• Open longitudinally from one end to the other (opposite the tumor)
• If a portion of the stomach is included, open along the greater
curvature in continuity with the esophageal cut
• Dissect the periesophageal fat and look for lymph nodes
• Divide into three portions: adjacent, proximal & distal to the tumor
• Paint the surgical specimens with ink after the specimens are fixed

Description
• Length & circumference of specimen; proximal stomach included
(indicate length along lesser & greater curvature)
• Tumor: size, appearance, circumferentially, depth of invasion;
extension into stomach, distance from both lines of resection
• Mucosa: Appearance of non-neoplastic mucosa; recognizable
esophageal mucosa distal to tumor evidence of Barrett esophagus,
lumen dilated proximal to tumor
• Wall: thickened, Varices
• Stomach, if present: features of cardioesophageal junction and
gastric mucosa
• Lymph nodes: no, size of largest; appear grossly involved by tumor

• Tumor: 4 longitudinal sections,1 including a portion of non-neoplastic
mucosa proximal to tumor & another portion distal to tumor
• Non-neoplastic mucosa: 2-3 transverse sections, at different
distances from tumor edge, proximally and/or distally, depending on
location of tumor
• Stomach, if present: two sections, one including gastroesophageal
junction
• Proximal and distal line of resection
• Lymph nodes: Adjacent to tumor, Proximal to tumor, Distal to tumor

Section should always be taken perpendicular to the direction of mucosal folds

Stomach – Gastrectomy for tumor
Procedure
• Open the specimen along the greater curvature (unless the lesion is
in this location; if it is, open the specimen along the lesser curvature)
• Dissect the lymph node groups
• If a splenectomy is included, dissect the hilar lymph nodes, measure
and weigh the spleen,
• Paint the surgical margins
• In general, take the sections perpendicular to the directions of the
mucosal folds
• If a thorough mapping of the mucosal abnormalities is desired, the
entire specimen can be submitted or the Swiss roll technique can be
used

Description
• Type of resection(total/subtotal); length of curvatures & duodenal cuff
• Tumor characteristics: Location, size, shape, depth of invasion;
presence of serosal involvement; blood vessel invasion; extension
into duodenum; distance from both lines of resection
• Appearance of non-neoplastic mucosa
• Tumor: 4 sections through wall & including tumor border and
adjacent mucosa
• Non-neoplastic mucosa: mid stomach, two sections
• PRM along lesser & greater curvature: two sections each
• DRM (along pylorus & duodenum): two sections
• Spleen and Pancreas, if present
• Lymph nodes

If mapping of mucosal abnormalities are desired such as in metaplasia, dysplasia a
swiss roll technique can be used, where mucosa alone can be shaved off from
underlying muscle layer and rolled up

Stomach – Gastrectomy for ulcer
Description
• Type of resection; length of greater, lesser curvature & duodenal cuff
• Ulcer characteristics: location, size, depth, shape and color of edges;
perforation at ulcer base; appearance of serosa.
• Appearance of uninvolved mucosa: atrophy, edema, hemorrhage
• Ulcer: at least four sections
• Lesser curvature: two sections cut from proximal margin of excision
• Greater curvature: two sections cut from proximal margin of excision
• Pylorus and duodenum: two sections, including distal line of resection
• Other lesions, if present
• Lymph nodes: up to three sections

Large bowel: Colectomy for nontumoral
condition
Procedure
• Sample lymph nodes & remove the mesentery while the specimen
is fresh
• Open the bowel longitudinally and fix overnight in formalin
• Take the sections perpendicular to the direction of the mucosal folds

Description
• Part of bowel removed, length of specimen & amount of mesentery
• Mucosa: type of lesions, extent, ulceration (linear or transverse),
depth, pseudopolyps, hemorrhage, fissures
• Wall: thickening (focal or diffuse), atrophy, fibrosis, necrosis
• Serosa: fibrin, pus, fibrosis, adherence of mesentery
• Diverticula: number, size, location in relation to teniae, content,
evidence of inflammation, hemorrhage or perforation
• As many as necessary to sample abnormal areas
• Proximal and distal lines of resection in cases of colitis
• Appendix, if included in specimen

Large bowel – colectomy for tumor
• Tumor: at least three sections (extending through the entire wall)
• Representative section of subserosal connective tissue, fat, and
blood vessel around tumor
• Other lesions of bowel
• Proximal and distal line of resection
• Bowel between tumor and distal line of resection (halfway or 5 cm,
whichever suits the case)
• Appendix, if included in specimen
• Lymph nodes: Around tumor, Distal to tumor, Proximal to tumor
• In abdominoperineal resections: anorectal junction

LYMPH NODES ACCORDING TO
TOPOGRAPHICAL SITES

Polypectomy
Procedure
• Measure the diameter of the head & length of the stalk
• For polyps with a short stalk or no stalk, identify the surgical section
and cut in half longitudinally.
• For polyps with a long stalk (≥1), cut a cross-section of the stalk near
the surgical margin and then cut the polyp longitudinally.
Description
• Dimensions of polyp; diameter of head and length of stalk
• Polyp sessile/pedunculated, Ulcerated, surface: smooth/papillary
• One longitudinal section(including surgical margin in polyps with
short stalk or no stalk)
• One cross-section of base of stalk (in polyps with long stalk).

Small bowel – excision
Procedure
• Cut longitudinally through the antimesenteric border & fix overnight
Description
• Length and diameter of specimen, Wall: thickness, abnormalities
• Mucosa: appearance; edema, hemorrhage,ulcerations, tumor (size,
location, circumferential involvement, depth of invasion)
• Serosa: fibrosis, peritonitis, adhesions
• Lymph nodes: size and appearance
• Mesentery; mesenteric blood vessels
• Depends on pathology present
• In cases of infarct: several cross-sections of mesenteric vessels

Appendix
Procedure
• Measure organ (length and greatest diameter)
• Divide specimen in two by cutting a cross-section 2 cm from tip
• Cut cross-sections of proximal fragment at 5 mm intervals
• Divide distal fragment in two by a longitudinal cut
Description
• Length and greatest diameter
• External surface: fibrin, pus, hyperemia
• Perforation, condition of mesentery
• Wall: any localized lesions
• Mucosa: hyperemic, ulcerated
• Lumen: obliterated, dilated, Content: fecaliths, stones

•Proximal 1/3 close to surgical margin: one cross-section.
•Mid one-third: one cross-section
•Distal one-third: one longitudinal section
•If tumor is present in the specimen, paint the surgical margin with India
ink and take an additional section from it

Several types of mastectomy procedures
• Halsted type radical mastectomy
• Modified radical mastectomy (also known as extended simple
mastectomy & total mastectomy)
• Simple mastectomy
• Subcutaneous mastectomy
• Tylectomy (lumpectomy, excisional biopsy)

Breast : Lumpectomy / Excisional biopsy
Procedure :
– Measure the specimen and orient
– Section specimen: if specimen is ≤ 3cm, cut 3–4 mm slices;
– if it is larger, bisect specimen transversely, place cut surface down, and
take sagittal blocks through superior and inferior portions
Description :
• Dimensions and consistency of specimen
• Appearance of cut sections: fibrosis, cysts, calcification, tumor(size,
color, borders, consistency, distance from surgical margins)
• Small specimens: submit in their entirety (up to five cassettes)
• Larger specimens: thorough sampling should be done
• This include any grossly visible lesions & inked surgical margins

Procedure & Description
• Side, type of mastectomy
• Orient specimen and take dimensions
• Structures included in specimen: skin, nipple, breast, major and minor
pectoralis muscles, fascia, axillary tissue
Features of external appearance:
• Shape and color of skin
• Location and extent of skin changes
• Appearance of nipple and areola
• Location of lesions
Features of cross-sections:
• Mass: Quadrant, depth beneath skin, size, shape, consistency,
necrosis, hemorrhage, calcification, relation to skin, muscle, fascia
• Lymph node - No, size, locations of nodes with grossly evident
tumor
Mastectomy

Breast:
• Take three sections of tumor; sample all lesions noted grossly and
least one section from each quadrant
• Pectoralis major muscle (in radical mastectomies): take one section
from any grossly abnormal area
• Nipple areolar complex- one section
Lymph nodes:
• All identified nodes should be processed. Small nodes are submitted
entirely; nodes >0.5cm sliced.
• If the axillary fat is grossly involved, a representative section should
be taken. Label in the following order:
Radical mastectomy: • Low, Mid & High axillary lymph nodes
. • Interpectoral nodes
Modified radical mastectomy: • Lower half • Upper half

Kidney – nephrectomy for nontumoral condition
Procedure
• Measure and weigh the organ
• Cut the kidney sagittally, strip the capsule, and carefully open the
pelvis, calyces, and ureter
• Take photographs & identify in one of them the sites of the sections
to be taken
• If stones are present, submit for chemical analysis, if indicated

Description
• Weight and size of kidney
• Capsule: amount of pericapsular tissue, thickness, adherence
• External surface: smooth scars; cysts( no, size, location, content)
• Cortex: color, width; striations apparent and orderly
• Medulla: color, width; medullary rays apparent and orderly
• Pelvis: size; dilated blunting of calyces, Stones
• Ureter: diameter, length, evidence of dilation or stricture
• Renal artery and vein; appearance

• Kidney: Three sections, each including cortex and medulla
• Pelvis: two sections
• Ureter

Kidney – Nephrectomy for tumor
Procedure
• Cut the kidney sagittally, strip the capsule, and carefully open the
pelvis, calyces, and ureter
Description
• Weight and dimensions of specimen; length & diameter of ureter
• Tumor characteristics: size, shape, location, extent, homogenicity,
necrosis, hemorrhage; invasion of capsule, perirenal tissues,
calyces, pelvis, and renal vein
• Uninvolved kidney: external surface, cortex, medulla; any additional
focal lesions
• Pelvis: dilated blunting of calyces,Stones
• Presence, number, size, and appearance of perirenal lymph nodes

Tumor:
• RCC: minimum of 3 sections (including one with adjacent kidney);
• Pediatric tumors: minimum of one section/cm of tumor diameter;
• Carcinoma of renal pelvis: minimum of 3 sections with adjacent
pelvis and/or renal parenchyma
• Kidney not involved by tumor: two sections
• Pelvis: one section in cases of RCC or pediatric tumors; two
sections in cases of carcinoma of renal pelvis
• Renal artery and vein- one section
• Ureter: one section in cases of RCC or pediatric tumors; one
section/cm of ureter resected (and any abnormal-looking areas) in
cases of carcinoma of renal pelvis
• Lymph nodes, if present

Bladder – cystectomy
Two options are available for the dissection:
• Open with scissors in a Y shape through the anterior wall and fix
overnight in formalin
• Fill with formalin, fix overnight and divide into anterior and posterior
halves by first cutting the lateral bladder walls and then sectioning
the prostate, beginning at the bladder neck and being careful to
make the cut through the urethra.
Description
• Size of bladder; length of ureters; other organs present
• Tumor characteristics: size , location, extent of invasion, shape
(papillary, ulcerated); multifocal lesions?
• Appearance of non-neoplastic mucosa; thickness of bladder wall
away from tumor

• Tumor: At least three sections, through bladder wall
• Bladder neck: one section
• Trigone: two sections
• Anterior wall: two sections
• Posterior wall: two sections
• Dome: two sections
• Any other abnormal-looking area in bladder mucosa
• Ureteral orifices, including intramural portion
• Ureteral proximal margins
• In males: prostate (two sections from each quadrant) and seminal
vesicles (one section from each).
• Other organs present
• Perivesical lymph nodes, if any

Prostate gland:Transurethral resection(TUR)
Procedure
– Examine all the fragments: Ca prostate is often yellow and/or
hard; submit for histology chips with these gross characteristics
Description
– Weight of specimen
– Size, shape, and color of chips
– All of specimen until four cassettes filled
– If an excess, one additional cassette for each additional 10 g of
tissue
• If carcinoma is identified microscopically and then remainder of the
tissue should be processed in its entirety, regardless of amount

Prostate gland – suprapubic prostatectomy
for nodular hyperplasia
Procedure
Step section the specimen into 3 mm slices
Examine each slice carefully for areas suspicious of carcinoma
Description
• Weight of specimen
• Shape, color, and consistency
• Presence of hyperplastic nodules, cysts, calculi, areas suspicious of
carcinoma
• Left lobe: three sections
• Right lobe: three sections
• Middle lobe: one section

Prostate gland: Radical prostatectomy for tumor
Procedure
• Orient the specimen and paint the surgical margins with India ink
• Shave the vasa deferentia, bladder neck and apical margins
• Serially section the prostate at 2–3 mm intervals from apex to base
• Lay the individual slices sequentially and examine them carefully.
• Use the cross-section of the urethra as a landmark
• Take photographs of the slices & mark the site of the sections taken
Description
• Weight and dimensions of specimen
• Organs present: Prostate, Urethra, s. vesicles, vas, lymph nodes
• Prostate: tumor (location in lobes, size, color, borders, capsular and
periprostatic extension)
• Urethra: patent? impinged by tumor?
• Seminal vesicles: involved by tumor?

• Vasa deferentia margin
• Proximal (bladder neck) margin & distal (apical) margin
• Seminal vesicles: proximal, mid & distal portions from each side
• Prostate: The individual slices can be submitted in toto by cutting the
slices to fit the standard cassette (right and left halves & anterior and
posterior quadrants)

Penis – Penectomy
Procedure :
• Introduce a catheter through the urethra and fix overnight at 4°C
• Paint the surgical margins (including the urethra) with India ink
• Cut longitudinally through the center: it should cut the urethra in two
Description
• Type of operation: partial, total, scrotal skin, testicles, inguinal nodes
• Length and diameter of specimen
• Tumor: location in relations to glans, prepuce, skin, and urethra;
size, color, borders, depth of invasion
• Glans penis & urethra: invaded by tumor?.
• Tumor: three sections
• Glans penis and urethra
• Surgical margin (including urethra)

Testicle – orchidectomy
Procedure
• Open the tunica vaginalis and cut the testicle sagittally
• Take photographs and identify the sites of the sections to be taken
• Cut serial slices of each testicles, perpendicular to the original section
• Cut the epididymis longitudinally throughout its entire length
• Make several cross-sections of the spermatic cord at several levels
Description
• Weight and dimensions of testicle
• Length of spermatic cord
• Features of tumor: size, color; consistency; homogeneity or lack of it;
presence of cysts, necrosis, hemorrhage, bone, or cartilage; tumor
extension to tunica albuginea, epididymis, cord, and other structures
• Features of non-neoplastic testicle: atrophy? fibrosis? Nodules?
• Features of rete testis and epididymis

• Tumor: At least 3 sections or one section/cm of tumor,
• At least one of which should include some uninvolved testicle.
• Most of the sections should include tunica albuginea.
• Always submit sections from hemorrhagic or necrotic areas of
tumor, as well as from solid or fleshy areas.
• Uninvolved testicle: two sections
• Epididymis
• Spermatic cord and surrounding soft tissue at a point about 1 cm
from testicle: one cross-section
• Spermatic cord and surrounding soft tissue at line of resection: one
cross-section

Uterus – cervical biopsy
• Procedure :
• Do not cut the specimen unless the individual pieces are ≥ 4 mm
• It is essential that all of the tissue received be processed
• Always carefully search the container and the underside of the lid
• Description :
• Number of pieces received, shape and color
• Measurement in aggregate
• Epithelial erosions? irregularity in epithelial thickness?
• Any evidence of tumors or cysts?
• Sections for histology :
• Submit the material in its entirety
• If specimens are received with a specific identification, label and
submit them separately

Cervical cone Biopsy
Procedure
• Specimen should be received intact with a suture identifying the 12
o’clock position
• Open the specimen by cutting longitudinally along12 o’clock position.
• Paint surgical margins with India ink
• Cut the entire cervix by making parallel sections, 2–3 mm apart,
starting at the 12 o’clock position and moving clockwise.
• Sections should be taken in such a way that the epithelium is
present in each section;
Description :
• Size and shape of cone; complete cast of cervix or fragmented?
• Epithelium: color, presence of irregularities, erosions, masses (size,
shape, location), cysts (size, content)

• All of the tissue must be submitted (except for trimming of the stroma)
• If the cone has been oriented to the 12 o’clock position, identify
separately:
•Sections from 12 to 3 o’clock

Uterus – hysterectomy
Procedure: (For Non-malignant lesion)
• Measure and weigh the specimen
• Open it through both lateral walls, from the cervix to the uterine
cornua
• Make a mark as to which half is anterior
• Make additional cuts through any large mass in the wall
• Make parallel sections through each half, about 1 cm apart
• Make several sections of the cervix along the endocervical canal
• Make at least one cross section of every myoma present, larger
myomas need additional cuts
• If tubes and/or ovary accompanies the specimen, follow instructions
for these organs

Description
• Type of hysterectomy: total, radical with salpingo-oophorectomy
• Shape of uterus: deformed, subserosal bulges
• Serosa: fibrous adhesions
• Wall: thickness, abnormalities
• Endometrium: appearance; thickness; polyps(size, shape); cysts
• Cervix: Ectoocervix, squamocolumnar junction, endocervical canal;
• Myomas: number, location; size; sessile /pedunculated,hemorrhage,
necrosis, or calcification

• Corpus: at least two sections taken close to fundus and including
endometrium, good portion of myometrium and, if thickness permits,
serosa;
• Additional sections from any grossly abnormal areas
• Myomas: at least one section per myoma, up to three; sections from
any grossly abnormal area (e.g. soft, fleshy, necrotic, cystic)
• Cervix: one section from anterior half and one from posterior half
• Cervical or endometrial polyps: to be submitted in entirety unless
extremely large

Hysterectomy For Malignant Tumours
Description
• Tumor: Location, size, appearance, color, extent of endometrial
extensions, presence of myometrial, serosal, parametrial (soft
tissue), venous, cervical, or tubal extension
• Three sections, one of which should be through area of deepest
invasion and be complete sections from surface of endometrium
through serosa
• Two sections from non-neoplastic endometrium;
• Soft tissue from left and eighth parametria
• If no obvious tumor present, sample entire endometrium by making
parallel sections, 2–3 mm apart,

Hysterectomy for cervical carcinoma
• Amputate the cervix from the corpus about 2.5 cm above the external
os
• Open the cervix with scissors through the endocervical canal at the
12 o’clock position
• Cervix: all tissue is submitted and identified separately from 1-12
o’clock
• Vaginal cuff (entire line of resection)
• Left soft tissue (for invasive cases only)
• Right soft tissue (for invasive cases only)
• Rest of uterus:
• Ovaries and tubes:
• Lymph nodes,

Ovary – oophorectomy
Description
• Size and shape; weight, if enlarged
• Capsule: Thickened, adhesions, hemorrhage or rupture
• Cut section: character of cortex, medulla, and hilum; cysts (size and
content); corpus luteum, calcification, Hemorrhage
• Tumors: size; external appearance: smooth or papillary, solid or
cystic,content of cystic masses; hemorrhage, necrosis, or
calcification.
For incidental oophorectomies: one sagittal section of each entire ovary,
For cysts: up to three sections of cyst wall (particularly from areas with
papillary appearance)
For tumors: 3 sections or one section/cm of tumor, whichever is greater
and one section of non-neoplastic ovary, if identifiable

Fallopian tubes – salpingectomy
Description
• Length and greatest diameter
• Serosa: fibrin? hemorrhage? fibrous adhesions to other organs?
• Wall: abnormally thick? Ruptured?
• Mucosa: atrophic? hyperplastic? appearance of fimbriated end
• Lumen: patent? dilated? content; diameter, if abnormally large
• Masses: size, appearance, invasion
• Cysts in paraovarian region: diameter, thickness of wall, content;
sessile or pedunculated?
• In cases of suspected ectopic pregnancy: embryo or placenta
identified? amount of hemorrhage; rupture?

For incidental tubes without gross abnormalities:
• Submit three cross-sections of each tube, taken from the proximal,
mid, and distal portions, submitted in the same cassette

For tubes with suspected ectopic pregnancy:
• Submit any tissue with gross appearance of products of conception.
• If none is grossly identified, submit several sections from the wall in
the area of hemorrhage as well as several from the intraluminal clot.
• If products of conception are not identified microscopically, submit
additional sections
For tubes with other lesions:
• As many as needed to adequately examine any abnormal areas.
• If tumor is present, at least three sections must be taken to include
grossly uninvolved mucosa

Tissue processing
• Designed to remove all extractable water from tissue, replacing it with
support medium that provide sufficient rigidity to enable sectioning of
the tissue without damage or distortion
• Factors affecting rate of processing:
• Agitation
• Heat
• Viscosity
• Pressure
• Stage of tissue processing:
• Fixation
• Dehydration
• Clearing
• Infiltration
• Embedding

Fixation
• Most commonly used reagent for the fixation is 10% neutral buffered
formalin
• The tissue bits should of the size of ~2x2cm & 2-3micrometer in
thickness.
• These bits are then placed in metal cassettes or capsules which are
then placed in the fixative.
• Tiny biopsies or small specimen can be wrapped in a filter paper and
then put in a cassette & fixed.
• To help in visualization of small fragment of tissue during embedding,
a few drops of 1% eosin is added

Dehydration
• Removal of free unbound water and aqueous fixatives from tissue
components.
• Dehydrating agents are strong hydrophilic, possessing strong polor
groups that interact with water by hydrogen binding.
• Dehydration should be accomplished slowly
• if concentration gradient is excessive, diffusion current across the cell
membrane may cause cell distortion.
• The various dehydrating agents used are ;
• Ethyl alcohol
• Ethano acetone
• Isopropyl alcohol
• Methanol
• Glycol

• Clearing agents act as an intermediary between dehydration &
infiltration solution
• They should be totally miscible with both the dehydrating fluid & the
embedding medium.
• It provide translucent appearance to the tissue
• Choice of a clearing agent depends upon the following :
• Rapid penetration of tissue
• Speedy removal of dehydrating agent
• Ease of removal by melted paraffin wax
• Minimal tissue damage
• Safety factors (low fammability, low toxicity)
• Cost and convenience
• Clearing agents: xylene, toluene, chloroform
Clearing

Infiltration & embedding
• Most popular infiltration and embedding medium
• It is a mixture of long chained hydrocarbons produced by mineral oils
• It permeates the tissue in liquid form and solidify rapidly when cooled
• The paraffin wax increases the optical differentiation, hardens the
tissue & helps in easy sectioning of the tissue.
• It is compatible to most routine, special stains as well as
immunohistochemistry protocols
• Alternative embedding media: Resin, gelatin and celloidin
Paraffin wax

Overnight processing schedule
Station Reagent Time Temp
1 10% formalin 1 hour 38 degree C
2 10% formalin 1 hour 38 degree C
3 50% alcohol 1 hour 38 degree C
6 90% alcohol 45 min 38 degree C
7 100 % alcohol 1 hour 38 degree C
8 100 % alcohol 45 min 38 degree C
9 Xylene 1 hour 60 degree C
10 Xylene 30 min 60 degree C
11 Paraffin 1 hour 60 degree C
12 Paraffin 1 hour 60 degree C

Microwave processors
• Microwave irradiation fixes the tissue
more effectively
• Large specimens which are soft to cut,
can be irradiated for a brief period &
then the tissue blocks are cut.
• The procedures of dehydration &
impregnation is acceralated.
• Tissue antigens are well preserved
following microwave irradiation.
• Microwave processing can be applied in
electron microscopy & IHC.

Embedding
• Embedding involves the enclosing of properly processed, correctly
oriented specimen in a support medium that provide external
support during microtomy.
• It must fill the matrix within the tissue, supporting cellular
components.
• It should provide elasticity and resisting distortion during sectioning

Embedding Centre
Molten wax
Cold plates
Wax dispenser
Wax flow adjuster
Heated chambersUsed lids
Hot surface

Embedding process
Dispense wax Align tissue Cool in place
Cassette on Top-up wax
Cool plate

Section Cutting (Microtomy)
• Microtomy is the means by which tissue is sectioned and attached
the surface for microscopic examination
• The instrument by which this is done is called as a Microtome.
• TYPES OF MICROTOMES:
• Sliding
• Rotary
• Rocking
• Freezing
• Base sledge
• ultramicrotome
• Microtome knives:
• Disposable blades
• Glass and diamond knives

Paraffin section cutting
• Flotation water bath
• Slide drying oven or hot plate
• Forceps, brush and teasing needles
• Clean slide
• Slide rack
• Ice tray
• Diamond pencil
Equipment required
Section adhesives
• Albumin, gelatin, starch
• Poly-L-lysine
• 3-aminopropyltriethoxysilane (APES)
• Charged or plus slides (permanent)

Rotary Microtome
• It is the most commonly used.
•Block holder moves up and down
while the knife remains fixed.
•Suitable for cutting of small
tissues
•Parts of a Microtome ( Rotary ) :
• Block holder
• Knife clamp screws
• Knife clamps
• Block adjustment
• Thickness gauge
• Angle of tilt adjustment
• Operating handle.

Staining (H & E)
Procedure
• Deparaffinize sections (2 changes of xylene, 10min each)
• Re-hydration (100%, 70%, 50% & 30%- 5min each)
• Wash briefly in distilled water.
• Stain in hematoxylin solution for 10 min.
• Wash in running tap water
• Differentiate in 1% acid alcohol for 30sec
• Wash running tap water for 1min.
• Bluing in 0.2% ammonia water for 30sec
• Counter-stain in eosin solution for 2 minute.
• Graded Dehydration (30%, 50%, 70% &100%- 5min each)
• Clearing with xylene (2 times- 5 min each)
• Mount with DPX

Mountants
 DPX ( Distrene Dibutyl phthalate Xylene ).
 Canada Balsam
 Colophonium resin
 Terpene resin

Histopathology specimen processing

Histopathology specimen processing

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