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MUST To KNOW in Histopathology 1
MUST To KNOW in Histopathology 1
Tissue Processing
Steps “FDCIETS SMoL”
1. Fixation
(Decalcification)
2. Dehydration
3. Clearing/Dealcoholization
4. Impregnation/Infiltration
5. Embedding/Casting/Blocking
6. Trimming
7. Sectioning/Microtomy
8. Staining
9. Mounting
10. Labeling (slides)
Fixation
st
Fixation 1 and most critical step
1’ aim: preserve cell (life-like)
2’ aim: harden & protect tissues
Most important: stabilization of proteins
pH 6.0-8.0
Temperature Room temp = Surgical specimen
0 to 4’C = EM and Histochem.
Microanatomical fixatives General microscopic study of tissues
a. 10% Formol saline
10-10 b. 10% Neutral Buffered Formalin
H-F c. Heidenhain’s SuSa
Z-Z d. Formol sublimate (formol corrosive)
B-B e. Zenker’s solution
f. Zenker-formol (Helly’s)
g. Bouin’s solution
h. Brasil’s solution
Cytological Fixatives Specific parts of the cell
a. Nuclear fixatives: w/ glacial acetic acid – destroys mitochondria & golgi
bodies (pH ≤4.6)
b. Cytoplasmic fixatives: w/o glacial acetic acid
c. Histochemical fixatives: preserves chemical constituents
Nuclear fixatives “BFNCH”
Bouin’s
BFNCH Flemming’s w/ acetic acid
Newcomer’s
Carnoy’s
Heidenhain’s SuSa
Cytoplasmic fixatives “HORFF”
Helly’s
HORFF Orth’s
Regaud’s
Shortcut: “X-XhKhOhR”
SlideXyleneRemove coverslipXyleneK2MnO4Oxalic acid Restain
Shortcut: “Xi6B1DiCuCoFSMiBXM”
Broken slide Xylene Incubate 6 Butyl acetate + 1 Durofix Incubate
Cut film Cold H2O to float film & section Film w/ section mount
incubate butyl acetate xylene mount
Ringing Sealing the margins of the coverslip
Prevent escape/evaporation of fluid
Immobilize the coverslip
Prevent sticking of slides
a. Kronig cement = 2 parts paraffin + 4-9 parts colophonium resin
b. Durofix (cellulose adhesives)
Immunohistochemistry
Enzyme histochemistry Trypsin & protease = most commonly used
IgG Most commonly used antibody
Polyclonal Rabbits (1’) > Goat (2’) > Pig (3’) > Sheep (4’) > Horse (5’) > Guinea pig (6’)
Monoclonal Mice
Epithelial Tumor Markers
(+) CK 7 “LUBO” = paired
(-) CK 20 Lung
Uterus
Breast
Ovary
(+) CK 20 Stomach
(-) CK 7 Colon
(+) CK 7 Transitional cell carcinoma of the bladder
(+) CK 20 Mucinous ovarian tumor
(-) CK 7 HCC
(-) CK 20 RCC
SCC
Thyroid carcinoma
Prostatic adenocarcinoma
EMA (Epithelial membrane (+) carcinoma “BuLK” = paired
antigen) Breast
Lungs
Kidney
CEA Oncofetal antigen
GI carcinoma
Differentiates adenocarcinoma (+) & mesothelioma (-)
TTF-1 (Thyroid Differentiates lung adenocarcinoma & mesothelioma
Transcription Factor) (+): Thyroid, lung, neuroendocrine tumors
PSA Prostate cancer
Intermediate Filament Markers
Actin Smooth muscle
Base-sledge
Rotary
Rocking
Biconcave 120 mm Both sides are Paraffin Rotary
concave
Plane wedge 100 mm Both sides are Frozen sections Sliding
straight Hard, tough tissue specimen (paraffin) Base-sledge
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Carmine Chromatin stain
Best Carmine Carmine + Aluminum chloride = For glycogen
Mucicarmine Carmine + Aluminum hydroxide = For C. neoformans and mucin
Picrocarmine Carmine + Picric acid = for neuropathological studies
Duke’s staging for neoplasia One of the most frequently applied for staging individual tumors
of the rectum
Biopsy
Biopsy Excision and exam (living subject)
Preferred: perform the biopsy at the periphery of the tumor (advancing tumor
margin)
Types of Biopsy
Exfoliative cytology Desquamated cells
Sex hormonal status in females
Sex chromatin phenotype
Excisional biopsy Complete removal of a lesion
Most reliable
Incisional biopsy Removal of part of a lesion/small piece of tumor directly incising the tumor
capsule
Preferred for large tumors that can’t be excised completely
Needle biopsy Aspiration of fluid
Bite biopsy Small pcs of tumor are removed w/ special forceps
Cutaneous biopsy Skin fragments
Punch biopsy For specimens >2mm embed in a single paraffin block
Shave biopsy Curettage specimens
Wedge biopsy Specimen is subdivided w/ a razor blade
Marginal excision Shell-out end