Escolar Documentos
Profissional Documentos
Cultura Documentos
Dense CT Dermis
Capsules
Tendons
Stroma of cornea
Special Connective Tissues
Cartilage Hyaline = trachea
Fibrous = intervertebral discs
Elastic = ear, epiglottis
Bone Cancellous/spongy/trabecular = epiphysis/ends of long bones
Compact/cortical = diaphysis/shaft
Others Blood
Lymph
Hematopoietic tissues
Acid Fixative: Lead fixatives
mucopolysaccharides Stain: Alcian blue
Osteogenesis imperfect Brittle bone disease
Defective production of collagen
Deposits found in Connective Tissues (Eosinophilic)
Fibrin Early: yellow
Old: blue
Stains:
Mallory‟s PTAH
Lendrum‟s MSB
Fibrinoid Necrotizing vasculitis
Staining reactions identical to fibrin
Mixture of exudates & altered cytoplasmic constituents
Hyaline Degenerated collagen
Hypertension, atheroma, diabetic kidney
Stain: PAS
Amyloid TB, leprosy, osteomyelitis
Stains: “CoMT”
Congo Red
Metachromatic stain
Thioflavine
Muscle Tissues
Smooth Involuntary
Intestines, blood vessels
Skeletal Striated, voluntary
Skeletal muscles
Cardiac Striated, involuntary
Heart
Nervous Tissues
Early pregnancy
Quantitative Evaluation: Cytohormonal Maturation Index (CHMI)
CHMI MI = P/I/S
Pregnancy
Newborn (8 weeks)
Infancy (8 weeks-
puberty)
Late menopausal MI = 100/0/0 (no estrogen)
75 y.o. woman w/ MI = 0/20/80
estrogen therapy
Quality Assurance
3 copies/report 1. Doctor
2. Patient = original copy
3. File
Reports Surgical pathology report
Cytopathology report
Autopsy report
Signatories Request forms = patient‟s doctor
Result forms = pathologist
Turnover of results Surgical pathology & cytology = 24 hrs
Frozen section = 5-15 mins
Autopsy report = 1 week (Autopsy procedure: 24 hrs)
Storage Specimen (tissue) = 1 month to 1 year
Tissue blocks (paraffin) = 3 to 10 years
Slides = indefinite
Suggested Guidelines for Record and Specimen Retention (Henry, 21st Ed.)
Records
Requisitions 2 years
QC 2 years
Instrument maintenance 2 years
BB QC 5 years
BB employee signatures 10 years
BB donor/recipient Indefinitely
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
Fixation 1st 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 General microscopic study of tissues
fixatives a. 10% Formol saline
b. 10% NBF
c. Heidenhain‟s SuSa
d. Formol sublimate (formol corrosive)
e. Zenker‟s solution
f. Zenker-formol (Helly‟s)
g. Bouin‟s solution
h. Brasil‟s solution
Cytological Specific parts of the cell
Fixatives 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
Flemming‟s w/ acetic acid
lec.mt 04 |Page | 269
Newcomer‟s
Carnoy‟s
Heidenhain‟s SuSa
Cytoplasmic “HORFF”
fixatives Helly‟s
Orth‟s
Regaud‟s
Flemming‟s w/o acetic acid
Formalin w/ post chroming
Histochemical “FANA”
fixatives 10% Formol saline
Absolute alcohol
Newcomer‟s fluid
Acetone
Aldehyde Fixatives
Formaldehyde Concentrated solutions should not be neutralized
(explosion)
Stock solution: 37-40%
Working solution: 10% (no buffer: unstable)
Formalin pigments:
a. Paraformaldehyde
- White crystalline precipitates
- Due to prolonged standing
- Removed by: 10% METOH/filtration
b. Acid formaldehyde hematin
- Brown/black granular deposits that may obscure
microscopic details
10% Formol saline CNS
10% NBF Best general tissue fixative
Best fixative for tissue containing iron granules
w/ double phosphate buffer
1 mm/hr = rate of tissue penetration
Formol-Corrosive w/ HgCl2
(formol sublimate)
Glutaraldehyde EM
Karnovsky‟s EM: electron histochemistry & electron
paraformaldehyde immunocytochemistry
- glutaraldehyde
Acrolein Mixture w/ formaldehyde/formaldehyde
Formol-calcium Lipids (frozen section)
Fixatives
Mercuric Chloride Tissue photography
For Trichrome stain (excellent)
Produce black granular deposits except SuSa
“BOSCHZZ”
a. B5 = for BM biopsies
b. Ohlmacher‟s
c. Schaudinn‟s
lec.mt 04 |Page | 270
d. Carnoy-Lebrun
e. Heidenhain‟s SuSa = (-) black pigments
f. Zenker‟s = recommended for trichrome staining
g. Zenker-formol (Helly‟s) = pituitary gland, BM, &
blood containing organs
Heidenhain‟s SuSa Su = sublimat (HgCl2)
Sa = saure (acid)
HgCl2 Shrinks tissues
G.HAc Swells tissues, counteracts HgCl2
De-zenkerization Removal of mercuric deposits
H2O I2 H2O Sodium thiosulfate H2O
Chromate “ROCK”
fixatives a. Regaud‟s (Moller‟s) = chromatin, mitochondria,
mitotic figures…
b. Orth‟s = for Rickettsia, tissue necrosis
c. Chromic acid = preserves CHO
d. K2CrO4 = mitochondria (if acidified, fixes
chromatin bodies & chromosomes but destroys
mitochondria)
Chromate Fine, yellow brown
pigments
Lead fixatives Used in 4% aqueous solution of basic lead acetate
For acid MPS and mucin
Picric acid Highly explosive when dry
fixatives Excessive yellow staining of tissues
Picrates Protein Ppt. (H2O soluble) Add 70%
ETOH Insoluble
Never wash in H2O before dehydration
For glycogen (excellent)
a. Bouin‟s = for embryos, Masson‟s trichrome stain,
glycogen
b. Brasil‟s alcoholic picroformol = less messy than
Bouin‟s, glycogen (excellent)
Glacial acetic acid Solidifies at 17‟C
Fixes & precipitates nucleoproteins, chromosomes,
& chromatin material
Most commonly combined w/ other fixatives
Alcoholic Disadvantage: polarization (glycogen granules
fixatives poles/ends of the cells)
“MEICAN”
a. Methanol = BM & blood smears
b. Ethanol = preserves but does not fix glycogen
(Disadv: polarization)
c. Isopropanol = for touch preparations
d. Carnoy‟s = most rapid (1-3 hrs) | for chromosomes
| Dx: rabies (acetone)
e. Alcoholic formalin (Gendre‟s) = sputum
f. Newcomer‟s = for MPS | nuclear & histochemical
lec.mt 04 |Page | 271
fixative
Osmium tetroxide Inhibits hematoxylin
(Osmic acid) Produce black precipitate crystals (osmium oxide)
For lipids
a. Flemming‟s = permanently fixes fat, for nuclear
structures (excellent)
- Fixative & decalcifying agent (chromic acid)
b. Flemming‟s w/o acetic acid = for mitochondria
Trichloroacetic Precipitates proteins
acid Swelling effect counteract shrinkage by other
fixatives
Weak decalcifying agent (softening effect)
Acetone Recommended for H2O-diffusible enzymes
(phosphatases, lipases)
Rabies
Heat fixation Bacteriologic smears
Microwave: 45-55‟C
Underheating: poor sectioning
Overheating (>65‟C): vacuolation, overstained
cytoplasm
2‟ fixation Placing an already fixed tissue in a 2nd fixative
Post- Primarily fixed tissue 2.5-3% K2CrO4 (mordant)
chromatization
Washing out Removing excess fixative
a. Tap H2O = remove excess chromates, formalin,
osmic acid (NOT Bouin‟s)
b. 50-70% alcohol = wash out excess picric acid
(Bouin‟s)
c. Alcoholic I2 = remove excess mercuric fixatives
EM fixatives Glutaraldehyde
PtCl3
PtCl3 – formalin (Zamboni‟s)
AuCl
Osmium tetroxide
10% NBF = acceptable but not recommended
Stains (EM) “PUL”
1. PTA = 1st general stain
2. Uranyl acetate = Best
3. Lead
Factors that Affect Fixation of Tissues
Retarded by:
Size & thickness
(+) Mucus Prevents complete penetration of fixative
Wash w/ NSS
(+) Fat Fatty tissues: cut in thin sections, fixed longer
(+) Blood Flush out w/ NSS fix
Cold temperature Inactivates enzymes
Shortcut: “X-XhKhOhR”
SlideXyleneRemove
coverslipXyleneK2MnO4Oxalic acid Restain
Broken slides 1. Mount the broken slide to another clean xylene-
moist slide w/ drop of mounting media
2. If replacement not possible, the section (if
intact) may be transferred to another slide:
Broken slide Xylene (rem. coverslip) incubate
(rem. mountant) 6 parts butyl acetate + 1 part
lec.mt 04 |Page | 282
durofix incubate (mixture film) Cut the
film around the section Cold H2O until the film &
section float off Film w/ section mount on a
clean slide incubate butyl acetate xylene
mount
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 Trypsin & protease = most commonly used
histochemistry
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 (+) carcinoma “BuLK” = paired
membrane Breast
antigen) Lungs
Kidney
CEA Oncofetal antigen
GI carcinoma
Differentiates adenocarcinoma (+) & mesothelioma
lec.mt 04 |Page | 283
(-)
TTF-1 (Thyroid Differentiates lung adenocarcinoma & mesothelioma
Transcription (+): Thyroid, lung, neuroendocrine tumors
Factor)
PSA Prostate cancer
Intermediate Filament Markers
Actin Smooth muscle
Skeletal muscle
Cardiac muscle
Vimentin Melanomas
Schwannomas
Desmin Leiomyoma (smooth muscle)
Rhabdomyosarcoma (skeletal muscle)
GFAP (Glial Astrocytoma
Fibrillary Acidic
Protein)
NF Neuroblastoma
(Neurofilament) Ganglioneuromas
Neuroma
Chemodectoma
Pheochromocytoma
S100 protein Low MW Ca2+-binding protein
CNS glial cells, Schwann cells
Neuroendocrine Markers
NSE (Neuron- Strong evidence of neural/neuroendocrine
specific enolase) differentiation
Others Chromogranin
Synaptophysin
Germ Cell tumor markers
HCG Synthesized by syncytiotrophoblasts
Choriocarcinoma
AFP Endodermal sinus tumors showing yolk sac
differentiation
PLAP (Placenta- Germinomas
like ALP)
Mesenchymal Tumor Markers
Myogenic tumors Myo-D1
Myoglobin
Myogenin
Fibrohistiocytic --
tumors
Vascular tumors Factor VII-related antigen
CD31
UEA: Ulex europaeus I
Melanomas --
Lymphomas LCA: Leukocyte common antigen (CD45)
Cell Proliferation Markers