HISTOPATHOLOGIC AND CYTOLOGIC TECHNIQUES - LECTURE

LESSON 1: PRE-ANALYTICAL FACTORS AND GROSS DESCRIPTION

MIDTERM | A.Y. 2022-2023 | PROF. DOREN VENUS OTOD

OUTLINE ○ requisition form and specimen container

I. Pre-Analytical Factors label must match
A. Pre-Analytic Fixation ● Unlabeled, mislabeled, and inappropriately
B. Specimen Reception identified specimens (last resort: DNA identification)
C. Specimen Accessioning ● Leaking specimen containers
II. Gross Examination ● Absent clinical data or history, and other necessary
A. Materials for Gross Examination
info
B. Specimen Categories
1. Specimen for Gross Description ○ one of the basis of the pathologist in
only reading or diagnosing the patient
2. Specimen Excluded from
Mandatory Submission C. SPECIMEN ACCESSIONING
III. Describing Specimens and Gross Description
IV. Sectioning SPECIMEN ACCESSIONING
V. Other Specimen Considerations ● 1st and most important step in histopath outside the
tissue processing procedures
I. PRE-ANALYTICAL FACTORS ○ the rest of the process will be affected, if
there is mislabeling
PRE-ANALYTICAL FACTORS
● Specimens are given a unique identification
● Factors being identified in order to know or have a
number that will identify each specimen for each
better result in performing or for the overall
patient
processing of the tissue
○ for easier retrieval of specimens of the
slides, as well as the tissue blocks
A. PRE-ANALYTIC FIXATION
○ It will depend on the protocol of the
hospital
PRE-ANALYTIC FIXATION
● Indicating codes may be used for the following:
● All parts to be examined must be initially fixed
Surgical, Autopsy, Cytology
before the actual gross examination
● Sample Format of Accession Number: Indicating
○ Gross examination must not be performed
Code-Year-ID Number of Specimen
to a fresh sample; the sample must be
○ E.g. #S94-12345
prefixed (preserved)
● Earlier fixation → better tissue preservation
● Improper fixation → impede tissue processing
● Observe proper tissue-to-fixative ratio (1:20)
○ Most common fixative in the laboratory is
10% formaldehyde
● 3-5mm thick tissues: fixed for 6-48 hrs
○ The longer the fixation time, the better
● 5mm thick tissues and large tissues (such as
● Avoid serial accessioning of similar specimen types
limbs): sectioned prior to fixation, or else, fixation
to reduce mix-up of specimens, and
will not be complete and may occur only at the
cross-contamination
periphery of the tissue
○ E.g. gastric biopsies are interspersed with
○ To allow proper fixation of the sample
other tissue types
B. SPECIMEN RECEPTION

SPECIMEN RECEPTION
● Specimens must be put in a container labeled with
patient’s name and specimen source/site, time
and date of collection, name of the surgeon, and
with pathology requisition form ○ Ideal setup → placed in between of the
same/similar specimen type to avoid
CRITERIA FOR REJECTION OF SPECIMEN confusion
● Discrepancies between requisition form and
specimen labels

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 II. GROSS EXAMINATION B. SPECIMEN CATEGORIES

GROSS EXAMINATION SPECIMEN CATEGORIES

● Consists of describing the specimen and placing
all or parts of it into a plastic cassette, in
preparation for tissue processing
● One of the basis of pathologists’ diagnosis
● Where the pathologist will choose a representation
of the tissues, most especially if the tissue is large
in size
● Involves selection of elements that appear to be of
clinical significance for histologic examination
A. MATERIALS FOR GROSS EXAMINATION
MATERIALS FOR GROSS EXAMINATION
1. Cutting Tools
○ Scissors
○ Forceps
○ Blade Holders
○ Blade
● Specimens only requiring transfer
from container to tissue cassette
● Ideally, it must be wrapped in a filter
A paper to ensure that the sample is
still present until the last processing
(infiltration)
● Examples
○ Endometrium
○ Breast core biopsies
○ Colonic series
● Specimens requiring transfer but
with standard sampling, counting,
weighing or slicing

B
● Examples
○ Small lipoma (made of fatty
tissues)
○ Small skin biopsy
○ Cervical LLETZ

● Simple dissection required with

sampling needing a low level of
diagnostic assessment and/or
The ideal chopping board is white — to clearly see the
C preparation
tissues ● Example
○ Prepuce
2. Gross Table or Gross Workstations ○ Gallbladder
○ Sink ○ Haemorrhoids
○ Tabletop ○ Appendix
○ Water supply ● Dissection and sampling required
needing a moderate level of
○ Irrigation system
assessment
○ Fume extraction/ventilation system ● Example
○ Water disposal unit D ○ Pigmented skin lesions
○ Large intestine (Crobin’s)
○ Skin with markers
○ Salivary gland tumour
● Specimens requiring complex
dissection and sampling methods
E ● Example
○ Thyroid (medullary Ca)
○ Breast cancer
○ Testis (seminoma)
○ Uterus (endomet, Ca)

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 ● Exempted for routine microscopic review:
○ Accessory digits
■ Excess finger/toe will be removed
and will be submitted to the
histopath lab for description only

○ Bunions (aka hallux valgus) & hammer

toes
■ Bony bump that forms at the joints
of the base of the big toe

○ Extraocular muscle from corrective

surgery

○ Inguinal hernia sacs in adult

○ Nasal bone & cartilage from rhinoplasty

○ Prosthetic breast implant

1. SPECIMENS FOR GROSS DESCRIPTION ONLY

SPECIMEN FOR GROSS DESCRIPTION ONLY

● There are specimens received in the histopath lab
but are not subjected for further processing; for
gross description only
● Are removed in the body not because of an
abnormality, but because of some instances where
the tissue/part must be removed in the body
● Sent in the lab for disposal
● Disease is not histologic level

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 ○ Prosthetic heart valves without
attached tissue
○ Tonsils and adenoids from children
■ not benign or malignant type
○ Umbilical hernia sacs in children
○ Varicose veins
● Foreskin (from circumcision)
● IUDs (Intrauterine Device)
○ no soft tissue attached
○ form of contraceptive for female
● Medical devices
○ catheters, gastrostomy tubes, stents, and
sutures
○ have not contributed to the patient’s
illness, injury, or death
● Middle ear ossicles
● Orthopedic hardware and other radiopaque
medical devices
○ provided that there is an alternative policy
for documentation for these types of
samples during surgical removal
● Placentas
○ do not meet with the criteria of the hospital
or laboratory for examination
● Rib segments or other tissues
○ removed only for purposes of gaining
surgical access but is not suggestive of
any form of malignancy
● Saphenous vein
○ harvested from a coronary artery bypass
● Skin or other normal tissue removed
○ such as cosmetic surgery, reconstructive
procedures, as long as there is no lesion
or any tumor or malignancies
● Therapeutic radioactive sources
● Normal toenails and fingernails
III. DESCRIBING SPECIMENS AND
GROSS DESCRIPTION

GROSS DESCRIPTION
1. Identify the specimen. Note and verify all
anatomical structures.
○ Type of organ/ tissue
○ Left or right
2. SPECIMENS EXCLUDED FROM 2. Identify orientation markers used by surgeons, if
MANDATORY SUBMISSION available
○ Inks - used to identify and orient the
SPECIMEN EXCLUDED FROM MANDATORY specimen’s components, distinguish
SUBMISSION samples, for embedding instructions
● Bone donated ○ Nicks - indicates laterality
○ usually submitted in the Bone Bank ○ Sutures - represented by LL: long lateral;
● Bone segments or SS - short superior
○ usually submitted in the Bone Bank
● Cataracts I. INKING
○ cloudy vision ● Purpose: to accurately and faithfully
● Dental appliances and teeth transmit information to allow accurate and
○ with no attached tissue reliable microscopic assessment of this
● Fat margin
○ removed by liposuction ○ Resection margins
● Foreign bodies ○ Embedding instructions
○ bullet or other medico-legal evidence; will ○ Orientation
be given directly to the law enforcement ○ Distinguish between samples
personnel ○ Identify the cut surface

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 ○ Acetic acid ○ Weight - of intact organs are rounded to
● Will set as an instruction for the embedding the nearest 0.1g
process ■ Only required for sample that are
○ Ink dots instruct the embedder to hyperplastic such as the uterus,
embed the tissues a certain way endocrine, breast, kidneys
■ India ink is used
IV. SECTIONING

SECTIONING
● Taking a representative sample of the tissue
● Indicate number of sections and blocks on the
gross description
○ Indicate also the number of cassettes used
● Specimen must fit easily into the standard cassette,
which measures 3 x 2.5 x 0.4cm
● Thickness: not more than 0.3cm to allow for
closing of cassette and fixative penetration
● Different color scheme used to identify ● When possible, edges of tissue should be squared
its orientation:
○ Blue (Superior) A. SECTIONING
○ Green (Inferior) ● Cut serially about 2mm thick to look
○ Black (Posterior or Deep) for small lesions. Lesions are then
○ Red (Medial) sampled for histologic exam. Filter
○ Yellow (Anterior) SMALL paper may be used in wrapping
SPECIMENS small sample
○ Orange (Lateral)
○ If 2 or 3 colors are needed, the
preferred color to be used is
black, blue, and orange
● Acetic Acid is used to remove the ink ● Cut an interval of 1cm thickness
(termed as breadloafing) to ensure
II. SUTURING that pathologic areas or tumoral
● The pathologist will identify the location of areas are identified
the suture

LARGE
SPECIMENS

“12 o’clock” marker

Cystoprostatectomy - an en bloc excision of

all cancer bearing tissues in the pelvis
“3 o’clock” marker including the bladder, the prostate

3. Describe all notable characteristics: LABELING

○ Type of specimen
○ Shape
○ Color
○ Texture/Consistency
○ Odor - noted only if the smell is obvious
○ Dimensions - (length, width, depth) are
rounded to nearest 1.0cm. For multiple
pieces, indicate size of the largest piece
● Paper tags are embedded in the cassette
● Labeled with accession number using (lead)
pencil. Markers and pens will dissolve upon
processing
● If printed, dot matrix must be used
● Original containers with specimen are saved until
case is signed out (backup evidence in cases of
discrepancies)

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Example of result
Sample Gross Description
V. OTHER SPECIMEN CONSIDERATIONS
OTHER SPECIMEN CONSIDERATIONS
1. Brain
○ Brain is fixed first before grossed
○ Tied at the Circle of Willis and suspended
■ Circle of Willis - circulation where
blood flows or the pathway of the
blood to supply oxygen to the
brain as well as the surrounding
structures
○ Must not touch side of container to avoid
deformity
○ Recommended: In 10% NBF (Neutral
Buffered Formalin) for 2-3 weeks
2. Colon Cancers
○ Polyps: base (the area where cautery
arteries are located) is always inked
■ Small polyps: bisected and
places in one cassette
■ Large polyps: sides are trimmed
away from the stalk, and stalk is
placed in a separate cassette
● different sides = placed
on same cassette; middle
parts = in different
cassettes
Large polyps
3. Dermatologic Specimen
○ Vertical orientation is always maintained
(using markers)
○ Punch biopsies are submitted whole
○ Tissues greater than 4mm are dissected
○ Skin ellipses: serially cut along the short
axis at 2 to 3 mm interval. The two most
distal sections or tips are submitted in two
separate cassettes. Remainder is
submitted in one or more cassettes
4. Eyes
○ Inject fixative first then gross
5. Hard Tissues
○ Wash in running water then immerse in
TSE softeners
○ Must undergo the process of
decalcification
6. Hollow Structures
○ Must be cut open longitudinally and fixed
with cottons inside (cottons soaked in
fixative)
○ Ex. stomach
7. Lymph Nodes
○ Most important component of tumor
resections because they are essential for
prognosis and planning therapeutic options
○ Should be received fresh and not
immersed in formalin
○ Node is bivalved, and entirely submitted
○ Sentinel lymph nodes: usually the first
lymph node to be involved during
metastasis. Entirely submitted. However,
large specimens may be bisected, and
submitted in one or two cassettes
8. Mastectomy
○ Note for weight, size of breast and axillary
dissection, skin ellipse, nipple scar, basal
margins
9. Pediatric Specimen
○ Additional processes such as IHC, flow
cytometry, cytogenetics and molecular
genetics is often done. These may require
fresh, frozen, or specially processed
tissues
10. Specimen with Tumor
○ Identify:
■ Site & size of tumor
■ Location & structure invaded by
tumor
■ Vascular invasion
■ Presence of lymph node
■ Distance from resection margin
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