Germ layers
Categories of tissues
Covering Epithelia
Simple squamous
Simple cuboidal
Simple columnar
Stratified squamous
Stratified columnar
Pseudostratified columnar
Glandular Epithelia
Exocrine glands
Endocrine glands
Pancreas
Merocrine
Apocrine
Holocrine
Connective Tissues
Connective tissue
Collagen
MUST TO KNOW IN HISTOPATHOLOGIC TECHNIQUES
1. Ectoderm
2. Mesoderm
3. Endoderm
1. Epithelial = 3 germ layers
2. Nervous = endoderm
3. Muscular = mesoderm
4. Connective = mesoderm
Bowman’s, endothelium, loop of Henle, alveoli
Ducts of glands, walls of thyroid follicles
Gallbladder (nonciliated)
Uterine tube (ciliated)
Skin (keratinized)
Vagina, esophagus, cervix (nonkeratinized)
Male urethra
Female reproductive tract (nonciliated)
Trachea (ciliated), Epididymis
w/ ducts
Tubular = stomach, uterus
Acinar/alveolar = pancreas, salivary glands
Tubuloacinar = prostate
Ductless
Exocrine = enzymes
Endocrine = hormones
No loss of cytoplasm
Goblet cells, sweat glands
w/ cytoplasmic loss
Distal portion is pinched off
Mammary glands
Disintegrating cell and its constituents released
Complete breakdown of cell
Sebaceous gland
Support
Framework
Cells are widely separated
Major ingredient of connective tissues
Stains: “VgMMAK”
Van Gieson
Mallory’s aniline blue
Masson’s trichrome
Alcian blue
Krajian’s aniline blue
General Connective Tissues
Loose CT
Wharton’s jelly (acid MPS)
BM (reticular)
Lymph node (reticular)
Embryo (mesenchyme)
Hypodermis
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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 mucopolysaccharides
Fixative: Lead fixatives
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
CNS
Brain, spinal cord
PNS
Peripheral nerves
Special receptors
Ear, eye, nose
Inflammation
Inflammation
Latin word: Inflammare (to set afire)
5 Cardinal Signs of Inflammation
1. Rubor
Redness
Blood flow  Injury
2. Calor
Heat

lec.mt 04 |Page | 256
3. Tumor
Swelling
4. Dolor
Pain
 Fluid extravasation
 Sensory nerves
Loss of function
Destruction of functional units
Acute inflammation
Vascular & exudative
---(Tissue)---> Microphages
Subchronic inflammation
Intergrade between acute & chronic
Chronic inflammation
Vascular & fibroblastic
---(Tissue)---> Macrophages
Inflammation according to Characterisics of Exudate
Serous inflammation
Serum/secretions from serosal mesothelial cells (3P’s)
Pulmonary TB
Fibrinous inflammation
Diphtheria, rheumatoid pericarditis
Early stage of pneumonia
Catarrhal inflammation
Hypersecretion of mucosa
Hemorrhagic inflammation Blood + exudates
Bacterial infections & other infections
Suppurative/purulent
inflammation
5. Functio laesa
Retrogressive Changes = Organ/Tissue smaller than normal
Developmental defects: AAHA
Aplasia
Incomplete/defective development of a tissue/organ
Ex. amastia (breast aplasia)
Atresia
Failure to form an opening
Hypoplasia
Failure of an organ to reach its matured size
Agenesia
Complete non-appearance of an organ
Atrophy
Physiologic atrophy
Natural
Thymus, brain, sex organs
Pathologic atrophy
Vascular atrophy
Pressure atrophy
Atrophy of disuse
Exhaustion atrophy
Endocrine atrophy
Brown atrophy
Lipofuscin
Progressive Changes = Organ/Tissue larger than normal
Hypertrophy
Increased tissue size due to increased cell size
Physiologic: ásize of uterus
Pathologic: Systemic hypertension
Hyperplasia
Increased tissue size due to increased cell number
Physiologic: Glandular proliferation of the female breast, ásize of uterus (preg.)
Pathologic: Skin warts due to HPV
Compensatory hyperplasia Ex. Enlargement of one kidney
Pathologic hyperplasia
Ex. Endometrial hyperplasia
Congenital hypertrophy
Phenytoin-induced
Degenerative Changes = Tissues have abnormalities
Metaplasia
Reversible
One adult cell type ↔ Another adult cell type
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Dysplasia
Anaplasia/
Dedifferentiation
Neoplasia/tumor
Oncology
Parts of a tumor
Types of tumor
Benign
Malignant
Leukemia
Lymphoma
Squamous cell papilloma
Squamous cell carcinoma
Hepatoma/
hepatocarcinoma
Melanoma/
melanocarcinoma
Ectopic pregnancy
Grading
Grade
I
II
III
IV
Staging
UICC
AJCS
TNM system
T
Reversible
One type of adult cell ↔ Changes in structural components
Irreversible
Criterion toward malignancy
Adult cell  More primitive cells (release tumor markers)
Continuous abnormal proliferation of cells w/o control (no purpose/function)
Ex. Leukemia
Study of neoplasm
Tumors
1. Parenchyma = active elements (tumor cells)
2. Stroma = CT framework
1. Capacity to produce death:
- Benign (Ex. mole)
- Malignant
2. Histologic characteristics:
- Medullary = cells (parenchyma) > supporting tissues (stroma)
- Scirrhous = supporting tissues (stroma) > cells (parenchyma)
“-oma”
“SaMe CarE”
“-sarcoma” = mesenchymal/CT
“-carcinoma” = epithelial tissues
Malignant
Benign
Malignant
Malignant
Malignant
Fallopian tube pregnancy
Grading
Aggressiveness/level of malignancy
Differentiated cells = resemble normal cells
Undifferentiated cells = younger cells
Broder’s Classification (Grading)
Differentiated Cells
Undifferentiated Cells
75-100%
0-25%
50-75%
25-50%
25-50%
50-75%
0-25%
75-100%
Staging
Size, extent of spread to lymph nodes, +/- metastases
TNM classification
Grading + staging
TNM System
Applicable to all forms of neoplasia
1’ tumor
#: denotes the size of tumor and its local extent
Tis = carcinoma in situ
Ta = non-invasive
Tx = cannot be evaluated
T0 = free of tumor
Treatment
Surgery
↓
↓
Radiation
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N
M
Teratomas
Apoptosis
Necrobiosis
Necrosis
Types of Necrosis
Coagulation necrosis
Liquefaction/colliquative
necrosis
Caseous/caseation necrosis
Gangrenous necrosis
Fat necrosis
Fatty degeneration
1’ changes
2’ changes
Algor mortis (1st)
T1 = lesion <2 cm (T1a = <0.5 cm | T1b = <1 cm | T1c = <2 cm)
T2 = lesion 2-5 cm (invasion in muscle)
T3 = skin and/or chest wall involved by invasion (T3a = deep muscle | T3b =
through organ)
T4 = tumor invasion/fixation (T4a = adjacent organ | T4b = fixation to bladder or
colonic wall, in breast, edema)
Regional lymph node involvement
High # denotes increasing extent of involvement
Nx = not evaluable
N0 = no axillary nodes involved
N1 = 1 mobile regional (axillary) node involved
N2 = multiple, mobile regional nodes involved
N3 = fixed regional lymph node involved
N4 = beyond regional lymph node involvement
Metastasis
M0 = no evidence of metastases
M1 = distant metastases are present
Mx = distant metastases not evaluable
Compound tumors
Greek: Monstrous tumors
May contain hair, teeth, bones
w/ heartbeat
Cellular Death
Programmed cell death (cellular suicide)
Physiologic cell death
Ex. normal sloughing off of skin cells
Pathologic cell death
Most common
Tombstone formation
“MyLKS”
Myocardium
Lungs
Kidneys
Spleen
Pus formation
Brain & spinal cord
Yellow, cheesy, crumbly material
TB, syphilis, tularemia, lymphogranuloma inguinale
Sulfide gas production
a. Dry gangrene = arterial occlusion
b. Wet gangrene = venous occlusion
Chalky white precipitates
Pancreatic degeneration
Liver
Somatic death
During somatic death
“CRC”: circulatory, respiratory, CNS failure
After somatic death
“ARLP DPA”: Algor mortis, Rigor mortis, Livor mortis, Postmortem clotting,
Dessication, Putrefaction, Autolysis
Postmortem cooling
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Cooling: 7’F/hr
Rigor mortis
Stiffening
1st: neck & head (2-3 hrs)
Persists for 3-4 days
Livor mortis
Lividity/suggillations
Purplish discoloration
After 10-12 hrs, it does not blanch on pressure or shift when the body is moved
Postmortem Lividity vs. Ecchymosis
Postmortem Lividity
Ecchymosis
Disappears on pressure (reappears when pressure is Opposite of postmortem lividity
released)
Oozing of blood (incision)
No oozing of blood (incision)
Postmortem Clot vs. Antemortem Clot
Postmortem Clot
Antemortem Clot
Settling of RBCs from plasma
Not readily detachable from the blood vessels
Chicken fat
No chicken fat
Currant jelly
No currant jelly
Assumes the shape of the vessel
Seldom assumes the shape of blood vessels
Rubbery consistency
Granular & friable
Dessication
Drying & wrinkling of the anterior chamber of the eye
Putrefaction
Invasion of intestinal microorganisms
Autolysis
Self digestion of cells
Lysosome: suicide sac of the cell, releases lysozyme
Organ Weights
Liver
1,100 – 1,600g
Brain
1,150 – 1,450g
Right lung
300-400g
Left lung
250-350g
Heart
250-300g
Spleen
60-300g
Thyroid
10-50g
Adrenals
4g or so each
Exfoliative Cytology
Exfoliative cytology
Desquamated cells
Pap smear stain method
Method of choice
Barr body
PAP smear
3 anatomical sites
1. Upper lateral third of the vagina
2. Ectocervix
(Stratified Squamous Epithelium)
--------------------------------T zone: detect cervical cancer-------------------------------(Simple Squamous Epithelium)
3. Endocervix
♫ 50% alcohol = All types
Fixation
50% alcohol = pleural & peritoneal
70% alcohol = sputum
95% alcohol = urine, bronchial & gastric
♫ Saccomanno’s fixative = 50% ETOH + 2% carbowax
Smear preparation
Fix immediately
2-3 slides/patient
a. streaking
b. spreading
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(2nd)
Fixing smear
Sputum
BAL
Jelly-like clots
GI specimen
Urine
Pap smear
Adhesive agents
3 primary materials used
for obtaining specimen for
Pap smear
Strawberry cervix
Shift to the left
Shift to the right
Shift to the midzone
Superficial cells
Intermediate cells
c. touch preparation/impression
d. pull-apart
Never touch the bottom of the fixative container
Equal parts of ethanol & ether = BEST (but highly flammable)
95% ethanol = commonly used
Spray fixatives = 1 ft away
Saccomanno’s fixative
3 specimen
(+) alveolar macrophage = sputum
(-) alveolar macrophage = saliva
P. carinii/P. jiroveci
Prevent by adding 300U heparin/100mL aspirate
If >½ hr delay of fixation  digestion of cells
Fasting: 8 hrs
50mL = cytology
10-15mL = UA
2nd urine = preferred
Dr. George Papanicolau (1940)
Smears: prepared by rotary motion
1. Mailing of specimens
- air drying after 2 hrs fixation
- glycerin technique
2. Egg albumin = not recommended as adhesive agent (intensifies stain by Light
Green)
Pooled human serum/plasma
Celloidin ether alcohol
Leuconostoc culture
1. Speculum
2. Ayer’s spatula = rotate 3600
3. Cytobrush = Os
T. vaginalis
Cells (Cervicovaginal Smears)
Parabasal | Intermediate | Superficial
↓ ----------Estrogen----------↑
45-50μm
Pyknotic nucleus
True acidophilia
Folds/curls on edges
a. Navicular cells = boat-shaped
Parabasal cells
15-30μm
Fried eggs w/ sunny side up
Endometrial cells
Groups of 3 or more
1-10 days after menst
Endocervical glandular cells Honeycomb appearance
Similar appearance to parabasal cells
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L. acidophilus
Pap’s stain: blue to lavender
Diabetic patients
Sish kebab appearance
Pear-shaped, blue-gray to blue-green
Pigs on a scruff appearance
Indicates T. vaginalis infection
Clue cells
Wrinkled prune appearance
w/ perinuclear halo
HPV (LSIL)
Formation of salt crystals
Doderlein bacillus
C. albicans
T. vaginalis
Leptothrix
G. vaginalis
Koilocytes
Ferning
Early pregnancy
Quantitative Evaluation: Cytohormonal Maturation Index (CHMI)
MI = P/I/S
CHMI
Pregnancy
Newborn (8 weeks)
Infancy (8 weeks-puberty)
Late menopausal
75 y.o. woman w/ estrogen
therapy
MI = 100/0/0 (no estrogen)
MI = 0/20/80
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 records Indefinitely
Reports
Clinical pathology lab
2 years
reports
Surgical pathology (and
10 years
BM) reports
Cytogenetics reports
20 years
Autopsy forensic reports
Indefinitely
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Specimens
Serum/other body fluids
Blood smears (routine)
Microbiology smears
BB donor/recipient
specimens
Pathology/BM slides
Pathology blocks
Cytogenetic slides
Cytogenetics diagnostic
images
Forensic Cases
Body fluids
Tissue for toxicology
Wet tissues
Paraffin blocks
Slides
Reports
Gross photographs/
negatives
Dried blood films
Frozen tissue for DNA
Autopsy
Types of autopsy
Preliminaries for PME
48 hours
7 days
7 days
7 days post-transfusion
10 years
10 years
3 years
20 years
1 year
1 year
3 years
Indefinitely
Indefinitely
Indefinitely
Indefinitely
Indefinitely
indefinitely
Autopsy (Postmortem Examination)
Gold standard for confirmation of a medical disease
Wherever scientific medicine of high quality is practiced, postmortem exams
are performed
Whenever a conscientious physician knows why he lost his patient, a
postmortem exam has been performed
Whenever criminal law is enforced
Whenever a death certificate shows accurately the causes of death & confirmed
medical diagnosis for the assembling of vital statistics, a postmortem has been
performed
Whenever there is medical research on the causes & nature of diseases such as
cancer, heart diseases & stroke, the investigative method is the postmortem
exam
An informed society requires a postmortem exam in human death for the good
of medical science, for the public’s health & for the future care of the living
patient
1. Complete autopsy
- Requires consent
- Complete examination of all organs, including the brain
2. Partial autopsy
- Part of the anatomy
3. Selective autopsy
- Restricted to at least a single organ (Ex. MI – heart)
1. Written consent from the next kin-abide by the extent or restrictions allowed
- Relative: oriented by the attending physician, not the pathologist
2. Death certificate (Old: Blue form | New: Blue border/frame)
- Signed by:
a. Physician
b. Pathologist (back): will sign when PME has been performed
3. Medical abstract or clinical data
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4. Medico-legal clearance
- Suspicious evidence of foul play
- Ex. physical injury
Other Uses of Death
Certificate
PME is permitted w/o
consent in the following
circumstances
If pathologist is not
available
The coroner has authority
in the following cases
Somatic death
Criteria for the
pronouncement of death
Burial & cremation purposes
Transport of body from hospital  funeral  cemetery
Medical insurance claiming
- If suicide: (-) insurance
- Acts of God (lightning, flood): (-) insurance
- Civil war: (-) insurance
1. When it is ordered by the police or coroner (NBI)
2. When it is necessary to complete the death certificate
3. When the deceased himself has given consent before he died (advanced
directive)
- Stipulate that in the event you will die, you will be giving out a consent for
autopsy
- Donate your organs for medical purposes or for transplantations
4. Deceased military personnel who dies in active services/training duty or
military services
The medico-legal examiner or the coroner has jurisdiction in medico-legal cases
& may authorize the pathologist to proceed w/ an autopsy
1. All natural deaths occurring in the hospital w/in 24 hrs of admission, unless
the case was attended by a private physician w/in 36 hrs of death
2. Newborns in the 1st 24 hrs of life
3. All injury cases, old or recent
4. All deaths due to unknown cases
5. All deaths due to suspicious cases
6. All abortion, whether self-induced or otherwise
7. All violent deaths
8. All accidental deaths
9. All sudden deaths
10. All cases w/o medical attendance w/in 36 hrs prior to the hour of death
11. All deaths due to drowning, hanging or strangulation (asphyxia)
12. All deaths due to shooting, stab wounds, burns, electricity, lightning,
tetanus, etc.
13. Homicides
14. All suicides
15. All poisoning
16. Stillborn = omission
17. Premature death
Death of an organism
Cessation of circulation & respiration (1960’s)
1. Advanced resuscitation techniques that are capable of reviving effectively
cases of clinical death
*Clinical death: cessation of heartbeat & respiration but the brain is still alive
but injured
2. Advance life-sustaining equipment capable of maintaining cardiovascular &
respiratory functions despite severe brain injury
3. Redefinition from cessation to irreversible cessation of cardiorespiratory
functions after resuscitation attempts
4. Brain death: cannot be revived anymore [National institute of neurological
diseases & stroke in the US (1977)]
- Clinically dead & dead are the same
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Criteria for brain death
American bar association &
national conference of
commission of uniform
state laws legislative
definition of death (1980)
American academy of
neurology
Postmortem changes
Technique of Virchow
Brain death: perpetual state of deep sleed
a. Coma (patient will not respond) & cerebral unresponsiveness
b. Apnea
c. Absent cephalic (brainstem) reflexes
d. Electrocerebral silence
criteria should be present for 30 mins at least 6 hrs after onset of coma & apnea
1. irreversible cessation of circulation & respiratory functions
2. Irreversible cessation of all functions of the entire brain, including the
brainstem is dead
Death:
1. Coma
2. Absence of the following:
- Motor response
- Pupillary response to light & pupils at mid-position
- Corneal reflexes
- Caloric responses
- Gag reflexes
- Coughing in response to tracheal suctioning
- Sucking & rooting reflexes
1. Algor mortis
- 1st demonstrable change after death is cooling of the body
- At room temp: 2’-2.5’F/hr (1st hr)
- 1.5-2’F/hr (next 12 hrs)
- 1’F/hr (next 12-18 hrs)
- As a rule, the body cools at 1.5’F/hr (50% of cases)
- Not a reliable indicator as to the time of death
2. Rigor mortis
- Rigidity of the body due to hardening of the skeletal muscles caused by a
series of physiochemical events after death
- ( formation of locking-chemical bodies
between actin & myosin
- This interlocking is fixed & produces rigor mortis w/o shortening of the
muscles
- Sets w/in 2 hrs after death (head & neck)
- Complete w/ 12 hrs
- Persists about 3-4 days
3. Livor mortis (postmortem lividity/hypostasis)
- Blood supply gravitates to the skin vessels w/c becomes toneless & dilate after
circulation ceases
- Becomes evident as early as 20 mins after death
- Fully evident w/in 4-8 hrs
- Tardien spots: petechiae
4. Postmortem clotting of blood
5. Discoloration of tissue
- Abdomen: green
- Formation of sulfur gases (bacteria)
6. Putrefaction
7. Dessication (mummification)
Techniques of Autopsy
Organs removed & dissected individually in the body
Most widely used metohd
lec.mt 04 |Page | 265
Technique of Rokitansky
Technique of Ghon
Technique of Letulle
Autopsy: Larynx  Rectum
Teasing/Dissociation
Crushing/squash
preparation
Smear preparation
Frozen section
Freezing of unfixed tissue
Freezing of fixed tissue
Formal (formol) calcium
Commonly used methods of
freezing
Staining methods
(frozen sections)
H&E
Freeze-drying
Freeze-substitution
Cold knife procedure
In-situ dissection in part combined w/ en bloc technique
♫ En bloc:
- By cavity
- Interrelated to each other
- Systemic dissection
- Ex. thoracic cavity (lungs, heart, diaphragm), respiratory system
En bloc technique
En masse technique
♫ En masse:
- All organs of thoracic, abdominal, & pelvic are removed at the same time
- Sweeping of all organs
Very popular, easy to do, convenient
Part of consent: organs should be retained completely or partially
Organs  set aside later
Body  undertaker of the body
Fresh Tissue Examination
Tissue specimen  Watchglass (isotonic solution)  BF/PC microscope
Tissue (<1mm)  Sandwich bet. 2 slides/coverslip  Vital stain
Spread lightly over a slide (wireloop/applicator)
Frozen Section
(-) Fixative
Best frozen section
To localize hydrolytic enzymes & other antigens
Derivative of formaldehyde
Fix at 4’C for 18 hrs
Liquid nitrogen = most rapid
Isopentane cooled by liquid nitrogen
CO2 gas
Aerosol sprays
“PATH”
Polychrome methylene blue
Alcoholic pinacyanol
Thionine
H & E = progressive, no decolorizer
a. Progressive
- w/o decolorizer
- for frozen sections
b. Regressive
- w/ decolorizer (acid-alcohol)
- for routine histology staining
w/o use of any chemical fixative
♫ Quenching: rapid freezing (-160’C)
♫ Sublimation: removal of H2O in the form of ice (-40’C) – vacuum
Similar to freeze drying but:
Frozen tissue  Rossman’s formula/1% acetone
Dehydrated in absolute alcohol
Any microtome
Uses CO2
Knife: -40 to -60’C
Tissue: 5 to -10’C
Environment: 0 to -10’C
lec.mt 04 |Page | 266
Cryostat procedure
(Cold microtome)
O.C.T. (Optimal Cutting
Temperature)
Steps
Fixation
pH
Temperature
Microanatomical fixatives
Cytological Fixatives
Nuclear fixatives
Cytoplasmic fixatives
Temperature: -18 to -20’C
Cryostat: refrigerated cabinet w/ rotary microtome
Best mounting media for cryostat sections
Tissue Processing
“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
1 and most critical step
1’ aim: preserve cell (life-like)
2’ aim: harden & protect tissues
Most important: stabilization of proteins
6.0-8.0
Room temp = Surgical specimen
0 to 4’C = EM and Histochem.
General microscopic study of tissues
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
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
“BFNCH”
Bouin’s
Flemming’s w/ acetic acid
Newcomer’s
Carnoy’s
Heidenhain’s SuSa
“HORFF”
Helly’s
Orth’s
Regaud’s
lec.mt 04 |Page | 267
Histochemical fixatives
Aldehyde Fixatives
Formaldehyde
10% Formol saline
10% NBF
Formol-Corrosive
(formol sublimate)
Glutaraldehyde
Karnovsky’s
paraformaldehydeglutaraldehyde
Acrolein
Formol-calcium
Mercuric Chloride
Heidenhain’s SuSa
HgCl2
G.HAc
De-zenkerization
Chromate fixatives
Flemming’s w/o acetic acid
Formalin w/ post chroming
“FANA”
10% Formol saline
Absolute alcohol
Newcomer’s fluid
Acetone
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
CNS
Best general tissue fixative
Best fixative for tissue containing iron granules
w/ double phosphate buffer
1 mm/hr = rate of tissue penetration
w/ HgCl2
EM
EM: electron histochemistry & electron immunocytochemistry
Mixture w/ formaldehyde/formaldehyde
Lipids (frozen section)
Fixatives
Tissue photography
For Trichrome stain (excellent)
Produce black granular deposits except SuSa
“BOSCHZZ”
a. B5 = for BM biopsies
b. Ohlmacher’s
c. Schaudinn’s
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
Su = sublimat (HgCl2)
Sa = saure (acid)
Shrinks tissues
Swells tissues, counteracts HgCl2
Removal of mercuric deposits
H2O  I2  H2O  Sodium thiosulfate  H2O
“ROCK”
a. Regaud’s (Moller’s) = chromatin, mitochondria, mitotic figures…
b. Orth’s = for Rickettsia, tissue necrosis
lec.mt 04 |Page | 268
Chromate pigments
Lead fixatives
Picric acid fixatives
Glacial acetic acid
Alcoholic fixatives
Osmium tetroxide
(Osmic acid)
Trichloroacetic acid
Acetone
Heat fixation
2’ fixation
Post-chromatization
Washing out
EM fixatives
Stains (EM)
c. Chromic acid = preserves CHO
d. K2CrO4 = mitochondria (if acidified, fixes chromatin bodies & chromosomes
but destroys mitochondria)
Fine, yellow brown
Used in 4% aqueous solution of basic lead acetate
For acid MPS and mucin
Highly explosive when dry
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)
Solidifies at 17’C
Fixes & precipitates nucleoproteins, chromosomes, & chromatin material
Most commonly combined w/ other fixatives
Disadvantage: polarization (glycogen granules  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 fixative
Inhibits hematoxylin
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
Precipitates proteins
Swelling effect  counteract shrinkage by other fixatives
Weak decalcifying agent (softening effect)
Recommended for H2O-diffusible enzymes (phosphatases, lipases)
Rabies
Bacteriologic smears
Microwave: 45-55’C
Underheating: poor sectioning
Overheating (>65’C): vacuolation, overstained cytoplasm
Placing an already fixed tissue in a 2nd fixative
Primarily fixed tissue  2.5-3% K2CrO4 (mordant)
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
Glutaraldehyde
PtCl3
PtCl3 – formalin (Zamboni’s)
AuCl
Osmium tetroxide
10% NBF = acceptable but not recommended
“PUL”
lec.mt 04 |Page | 269
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
Fatty tissues: cut in thin sections, fixed longer
Flush out w/ NSS  fix
Inactivates enzymes
(+) Fat
(+) Blood
Cold temperature
Enhanced by:
Size & thickness
Agitation
Automatic/mechanical tissue processing
Moderate heat
37-56’C
Principles and Precautions in Handling and Fixation of Specimens in General
Autopsy materials
Fixed ASAP
If not possible  mortuary refrigerator (4’C) or arterial embalming
Surgical specimens
Fixed ASAP
If not possible  refrigerate
If placed in NSS during
Autolysis may occur before fixation
operation
If tissues are refrigerated
Avoid slow freezing (ice crystal formation)
Repeated freezing & thawing  destroy organelles, release enzymes…
Not more than 5mm thick
Size of tissues
Except lung edema: 1-2 cm thick
20:1
Ratio of fixative to tissue
Except osmium tetroxide (expensive) = ratio is 5-10:1
50-100:1
Ratio of fixative to tissue in prolonged fixation (ex. museum preparation)
Avoid drying of small tissue To prevent: place in a petri dish w/ moistened filter paper
biopsies
Hollow organs
Stomach, intestines
Packed w/ cotton soaked fixative or completely opened before being immersed
in adequate fixing solution
Air-filled lungs
Float on fixative
To prevent: cover w/ several layers of gauze to maintain it under surface
Human brains
Suspended by a cord tied under the Circle of Willis to prevent flattening
Avoid Ringer’s lactate for washing out of blood  intravascular perfusion
Fixation time: 2 weeks
Eyes
Not dissected before fixation  tissue collapse & wrinkling (escape of vitreous
humor)
Inject formol-alcohol before immersing the organ in the fixative
Glycogen-containing tissues Do not use water
Glycogen is water-soluble
Hard tissues
Cervix, uterus, fibroids, hyperkeratotic skin, fingernails
Wash in running water overnight  immerse in 4% aqueous phenol for 1-3
days (Lendrum’s method)
Difficulties Encountered because of Improper Fixation
Problem
Cause
Failure to arrest early cell autolysis
Failure to fix immediately (tissue was allowed to dry
before fixing)
Insufficient fixative
lec.mt 04 |Page | 270
Removal of substances soluble in fixing agent
Wrong choice of fixative
Presence of artifact pigments on tissue sections
Incomplete washing of fixative
Tissues are soft & feather-like in consistency
Incomplete fixation
Loss/inactivation of enzymes needed for study
Wrong choice of fixative
Shrinkage & swelling of cells & tissue structure
Overfixation
Tissue blocks are brittle & hard
Prolonged fixation
♫ An incompletely fixed tissue may lead to improper & incomplete clearing & impregnation, and may later
prove to be a hindrance to normal sectioning & staining of specimen
Pigment
Acid formaldehyde hematin
Mercuric chloride pigment
Chromate pigment
Osmium tetroxide pigment
Crush artifact
20:1
37’C
55’C
RT (18-30’C)
24-48 hrs
Decalcifying agents
HNO3
5% Formic acid
HCl (Von Ebner’s)
EDTA
Ion exchange resins
Electrophoresis
Measuring extent of
decalcification
Post-Decalcification
Tissue softeners
Color
Brown/black granules
Removed by:
“SAKaL”
a. Saturated picric acid
b. Alcoholic KOH
c. Kardasewitsch method
d. Lillie’s method
Black granules
Alcoholic iodine
Fine, yellow brown
Acid-alcohol
Black precipitate crystals
Cold H2O
Intense eosinophilic staining at the center of the tissue (H & E)
Due to partial coagulation of partially fixed protein
Decalcification
Ratio of decalcifying agent to tissue
Impaired nuclear stain by Van Gieson’s stain
Tissue  Digestion (24-48 hrs)
Optimum temperature
Time
Acids
Chelating agents (EDTA/versene)
Ion exchange resins
Elec. ionization (electrophoresis)
Most common
a. Perenyi’s = tissue softener & decalcifying agent
b. Phloroglucin-HNO3 = most rapid
- Disadvantage: Yellow color on tissue (neutralize w/ sodium thiosulfate)
Both fixative & decalcifying agent
Best general decalcifying agent
For small pcs of bones & teeth
For small pcs of bones & teeth
For surface decalcification (HCl)
For EM, IHC, & enzyme staining
Hastens decalcification by removing calcium ions from formic acid-containing
decalcifying solutions
Ca2+ are attracted to negative electrode (cathode)
Physical method
Chemical method = CaOx test (routine) | Turbidity = (+) Ca2+
X-ray = most ideal, most sensitive, most reliable but very expensive
- X-ray paper = Kodak X-omat or Faxitron
Removal/neutralization of acid from the tissues after decalcification
Lithium carbonate or sodium bicarbonate solution
4% phenol
Molliflex = tissues appear swollen & soapy
2% HCl
lec.mt 04 |Page | 271
Dehydration
10:1
Ethanol
Methanol
Butanol
Denatured alcohol
Acetone
Dioxane
(Diethylene dioxide)
Tetrahydrofuran (THF)
Graupner’s method
Weiseberger’s method
Cellosolve
Triethyl phosphate
Additives to dehydrating
agents
Methods of determining
incomplete dehydration
Xylene (Xylol)
Toluene
Chloroform
Benzene
Methyl salicylate
Methyl benzoate
Cedarwood oil
Clove oil
CCl4
Aniline oil
Glycerin
Gum syrup
Others
1% HCl in 70% alcohol
Dehydration
Aim: To remove fixative & H2O
Ascending grades of alcohol (Start: 65%)
Embryonic & animal tissues: 30% ETOH
Ratio of dehydrating agent to tissue
Best dehydrating agent
Blood & tissue films
Plants & animals
Ethanol + methanol
Both fixative & dehydrating agent
Both dehydrating & clearing agents
Dehydration w/ dioxane
Ethylene glycol monoethyl ether
Combustible and toxic
-a. 4% phenol + 95% ETOH = softener
b. Anhydrous CuSO4 (Last ETOH bath)
- both dehydrating agent & indicator of H2O content of 100% ETOH
- (+) H2O = White  Blue
1. Anhydrous CuSO4 method
2. Xylene  Milky
Clearing
Most commonly used
Clearing time: ½ to 1 hr
Block size: <5mm
Substitute for xylene/benzene
Clearing time: 1-2 hrs
Not carcinogenic
Toxic fumes
Toxic to liver
For clearing tough tissues
Does not make tissue translucent but removes alcohol
For urgent biopsies
Minimum shrinkage
Aplastic anemia
For double embedding techniques
For CNS, smooth muscles, skin
Minimum shrinkage
Has tendency to become adulterated
Similar to chloroform but is cheaper
Disadvantage: similar to chloroform
For delicate tissues, embryos and insects
No dealcoholization but make the tissues clearer
Citrus fruits oil
Trichloroethane & petrol
Impregnation
lec.mt 04 |Page | 272
25:1
Medium
Paraffin
Paraffin filters
Paraffin oven
Ratio of infiltrating medium to tissue
Paraffin wax
Celloidin (collodion)
Gelatin = H2O soluble, not a wax
Plastic = EM
Introduced by Bütschlii
Not recommended for fatty tissues
Low MP = paraffin is soft
High MP = paraffin is hard
Manual: At least 4 changes of wax at 15mins interval
Filtration at 2’C above the MP of wax
Ex. Green’s no. 904 (coarse filter paper)
2-5’C above the melting point of wax (55-60’C)
>60’C = shrinkage & hardening of tissues
Substitutes for Paraffin Wax
Paraplast
56-57’C MP
More elastic & resilient
Bones & brain
Embeddol
56-58’C MP
Bioloid
Eyes
Tissue mat
Contains rubber
Ester wax
46-48’C MP
Soluble in 95% ETOH & clearing agent
Impregnation w/o prior clearing
H2O soluble wax
38-42’C/45-56’C MP
Mostly PEG
♫ Carbowax: most commonly used
- Does not require dehydration and clearing
- For enzyme histochemistry
Celloidin/Collodion
Purified form of nitrocellulose
Wet celloidin
Equal parts of ether & alcohol
Bones, brain, teeth sections
Dry celloidin
Gilson’s mixture: equal parts of chloroform & cedarwood oil
Whole eye sections
LVN
Another form of celloidin
Soluble in equal concentrations of ether & alcohol
Plastic/Resins
Epoxy (EPON™)
Polyester
Acrylic
Waterbath
6-10’C below the MP of wax (45-50’C)
Autotechnicon
Fixes, dehydrates, clears & infiltrates tissues
Constant tissue agitation
Elliott Bench-Type processor
Wax bath: 3’C above the MP of wax
Vacuum embedding
Wax impregnation under negative pressure (hasten removal of air bubbles)
Time is reduced from 25-75% of the normal time required for tissue processing
2-4’C above the MP of wax
(+) Odor in the clearing
Indicates that the paraffin wax should be changed
agent
Embedding
Orientation
Arranging in precise positions in the mold, microtome & slide
Oven
5-10’C above the MP of wax (Impregnation: 2-5’C above)
lec.mt 04 |Page | 273
Blocking-out Molds
Leuckhart’s embedding
mold
Compound embedding unit
Plastic embedding rings &
basse mold
Tissue Tek
Disposable embedding
molds
Celloidin/Nitrocellulose
method
Double embedding method
Cold knife procedure
Cryostat (Cold microtome)
Fixation
Freeze-drying
Algor mortis
Formol calcium fixation
Impregnation
Embedding
Flotation water bath
Trimming
Routine histopath. (Rotary)
Freezing
EM (Ultrathin)
Rocking/Cambridge
microtome
Rotary/Minot microtome
Sliding microtome
2 L-shaped strips
Adjustable
Several compartments
Special stainless steel base mold fitted w/ a plastic embedding ring (block
holder)
Warm plate
Cold plate (-5’C)
1. Peel-away: perfect even block w/o trimming
2. Plastic ice trays: ordinary refrigerators
3. Paper boats: cheap & easy to make
Bones, teeth, eyes
Bell jars: control evaporation
1st: celloidin
2nd: paraffin
Brain
Recall Temperatures
Knife = -40 to -60’C
Tissue = 5 to -10’C
Environment = 0 to -10’C
-18 to -20’C
Surgical specimen: room temp
HC & EM: 0-4’C
Quenching: -160’C
Sublimation: -40’C
7’F/hr (3.89’C/hr)
4’C
Manual = 2-5’C above MP of wax (55-60’C)
Automated = 3’C above MP of wax
Vacuum = 2-4’C above MP of wax
5-10’C above MP of wax
6-10’C below MP of wax (45-50’C)
Trimming
Removing excess wax after embedding
Ideal: four-sided prism/truncated pyramid
Microtomy
4-6μm
10-15μm
0.5μm
Trefall
Simplest
Minot
Most commonly used for paraffin embedded tissues
Adams
Most dangerous (movable knife)
a. Base-sledge
- For all forms of media
- Block: moving
- Knife: stationary
b. Standard sliding
- Block: stationary
- Knife: moving
lec.mt 04 |Page | 274
Vibrotome
Ultrathin microtome
Freezing microtome
Clearance angle
Bevel angle
Honing
Types of hones
Stropping
Natural dyes
Synthetic dyes
Chromophores
Auxochrome
Chromogen
Dye
Chromophores
Auxochromes
Dye modifiers
Van der Waals forces
Sudanophilia
Hematoxylin
For unfixed, unfrozen tissues
For enzyme demonstration
EM
Diamond knives or broken plate glass
Queckett
Knife to tissue block: 0-150 angle
27-320 angle
Removal of gross nicks
Heel to toe (Edge 1st)
a. Belgium yellow: Best
b. Arkansas
c. Fine carborundum: for badly nicked knives
Removal of gross burrs
Toe to heel (Edge last)
Paddle strop (horseleather)
- Mineral oil = not recommended
- Vegetable oil (castor oil) = applied into the back of the strop, not the surface
Staining
From plants & animals
a. Hematoxylin
b. Cochineal dyes: female Coccus cacti
c. Orcein: from lichens
d. Saffron
A.k.a. coal tar dyes
Derived from benzene & collectively known as aniline dyes
Greek: “color-bearer”
Coloring property
Greek: “increasers”
Dyeing property
Benzene + Chromophore
Imparts color temporarily
Chromogen + auxochrome
Imparts color to tissue almost permanently
a. Quinoid ring: Basic fuchsin
b. Azo groups: Congo red
c. Xanthene: Eosin
d. Quinone-imine group
- Oxazin: cresyl fast violet
- Thiazins: toluidine blue
Cationic auxochromes: amino group (NH3+)
Anionic auxochromes: hydroxyl (OH-) and carboxyl (COO-) groups
Attached on benzene ring
a. Ethyl group
b. Methyl group
c. Sulphonic acid
Alum hematoxylin
Tissue stained w/ fat or oil-soluble dyes
H & E Staining
Hematoxylin capechianum/ Hematoxylon campechianum
Nuclear/basic/1’ stain
Waldeyer: 1st to use hematoxylin
lec.mt 04 |Page | 275
Lake
Oxidizing agents
Alum hematoxylin
Iron hematoxylin
Tungsten hematoxylin
Copper hematoxylin
Eosin (Eosin Y)
Coplin jar
Slotted staining dishes
Metal/glass staining racks/
carriers
H & E staining steps
Hematoxylin ---(Ripening)---> Hematein (active coloring substance)
Tissue-Mordant-Dye complex
H2O2
HgO2 = Harris’
K2MnO4
Na perborate
Na iodate = Mayer’s, Ehrlich’s, Gill’s
Routine H & E = Red
Mordant: K Alum
“MEGDH”
Mayer’s = Na iodate (ripening agent)
Ehrlich’s = Na iodate (ripening agent)
Gill’s
Delafield’s
Harris’ = HgO2 (ripening agent)
Mordant = oxidizing/ripening agent = Iron
a. Weigert’s
- Mordant: FeCl3
- Weigert’s + Van Gieson’s = CT & E. histolytica
b. Heidenhain’s
- Mordant: Ferric ammonium sulfate
a. Mallory’s PTAH
- Mordant = sunlight/K+
- Stain fibrin
Spermatogenesis
Cytoplasmic/acidic/2’ stain
Counterstain
a. Eosin Y (Yellowish) = most commonly used
b. Eosin B (Bluish) = deep red
c. Ethyl eosin/Eosin S/Eosin alcohol soluble
Holds 5-9 slides
Holds 5-19 slides
Holds 10-30 slides
1. Xylol (2) = deparaffinization
2. Descending grade of alcohol = rehydration
3. H2O
4. Remove fixative artifact pigments after rehydration & before staining
5. Stain: Nucleus = light blue
6. H2O
7. Acid alcohol (differentiator): Nucleus = light blue
8. Ammonia water (blueing agent): Nucleus = blue
- NH4OH
- LiCO3
- Scott’s tap H2O
9. Wash
10. Stain: Eosin Y
11. Ascending grade of alcohol = dehydration
12. Xylene = dealcoholization/clearing
13. Mount & label
Nuclei: blue to blue black
Cytoplasm: pale pink
lec.mt 04 |Page | 276
Pap Smear staining
Benzidine
Acridine orange
Gentian violet
Congo red
Iodine
Malachite green
Janus green
Night blue
Victoria blue
Lysochromes
Adhesives
Mounting media
Stains on skin
Restaining of old sections
Broken slides
Hematoxylin = nuclear stain
OG-6 = cytoplasmic stain (mature superficial cells)
EA (Eosin Azure) = cytoplasmic stain (immature cells: parabasal/intermediate)
EA 65 = for body fluids
EA 36/50 = for gynecologic smears
Other Stains
Hgb
RNA (red fluorescence)
DNA (green fluorescence)
Crystal violet + methyl violet + dextrin
Elastic tissue, amyloid, myelin
Oldest stain
Ascaris eggs
Mitochondria (intravital stain)
Substitute for carbolfuchsin
Neuroglia
Oil soluble dyes
a. SBB = Black (most sensitive)
b. Sudan III = orange
c. Sudan IV (Scharlach R) = red
a. Mayer’s egg albumin = add thymol crystals (inhibit mold growth)
b. Dried albumin
c. Gelatin
d. Gelatin-formaldehyde
e. Starch paste
f. Plasma
g. Poly-L-lysine = IHC
h. 3-APES: 3-aminopropyltriethoxysilane = Best (cytology)
♫ 1.518 = refractive index of glass
1. Resinous media = contains xylene
- a. DPX = 1.532
- b. XAM = 1.52
- c. Canada balsam (Abus balsamea) = 1.524
- d. Clarite = 1.544
2. Aqueous media = for lipids (no xylene)
- Water = temporary mounting, low refractive index
- Glycerin jelly = 1.47 | standard mounting medium (fat stains)
- Gum Arabic (Farrant’s) = 1.43
- Apathy’s medium = 1.52
- Brun’s fluid = for frozen sections
Others:
- Permount
- HSR
- Clearmount
Remove by using 0.5% acid alcohol  tap water
Slide  Xylene (24hrs) or gently heat until mounting medium begins to bubble
 Remove coverslip  Section: Xylene (30mins)  H2O  0.5% K2MnO4
(5mins)  H2O  5% Oxalic acid (5 mins or ‘til decolorized)  H2O  Restain
Shortcut: “X-XhKhOhR”
SlideXyleneRemove coverslipXyleneK2MnO4Oxalic acid Restain
1. Mount the broken slide to another clean xylene-moist slide w/ drop of
lec.mt 04 |Page | 277
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 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
Ringing
Enzyme histochemistry
IgG
Polyclonal
Monoclonal
Epithelial Tumor Markers
(+) CK 7
(-) CK 20
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
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
Trypsin & protease = most commonly used
Most commonly used antibody
Rabbits (1’) > Goat (2’) > Pig (3’) > Sheep (4’) > Horse (5’) > Guinea pig (6’)
Mice
“LUBO” = paired
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
Skeletal muscle
lec.mt 04 |Page | 278
Vimentin
Desmin
GFAP (Glial Fibrillary Acidic
Protein)
NF (Neurofilament)
S100 protein
Neuroendocrine Markers
NSE (Neuron-specific
enolase)
Others
Cardiac muscle
Melanomas
Schwannomas
Leiomyoma (smooth muscle)
Rhabdomyosarcoma (skeletal muscle)
Astrocytoma
Neuroblastoma
Ganglioneuromas
Neuroma
Chemodectoma
Pheochromocytoma
Low MW Ca2+-binding protein
CNS glial cells, Schwann cells
Strong evidence of neural/neuroendocrine differentiation
Chromogranin
Synaptophysin
Germ Cell tumor markers
HCG
Synthesized by syncytiotrophoblasts
Choriocarcinoma
AFP
Endodermal sinus tumors showing yolk sac differentiation
PLAP (Placenta-like ALP)
Germinomas
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
Ki67
MIB-1: reference monoclonal antibody for Ki67 demonstration
PCNA
Proliferating cell nuclear antigen
Controls
Positive control
Known
Contains antigen in question
Negative control
Done using a parallel section from the tissue
Internal tissue control
A.k.a. “built-in control”
Contains the target antigen
Other Topics
Faults During Tissue Processing
Brittle/hard tissue
Clearing agent  Milky
Incomplete dehydration
lec.mt 04 |Page | 279
On trimming, tissue smells
of clearing agent
Tissue is opaque
Tissue shrinks away from
wax
Tissue is soft when block is
trimmed
Air holes on tissue
Wax appears crystalline
Paraffin block is moist &
crumbles
Sections fail to form
ribbons
Sections roll up on cutting…
adhere & get broken
against the knife edge
Ribbon is curved, crooked,
or uneven
Sections are compressed,
wrinkled or jammed
Sections are squashed
Hole in section
Sections of unequal
thickness
Sections adhere to knife or
other parts of the
microtome
Insufficient impregnation
Insufficient clearing
Insufficient dehydration
Incomplete clearing & impregnation
Incomplete fixation
Incomplete impregnation
Contaminated wax
Block not cooled rapidly enough
Insufficient paraffin impregnation
Surface & edges of block not parallel
Wax too hard
Knife tilted too much
Thick sections
Dull knife
Blunt knife
Dirty knife edge
Irregular knife edge
Edges of block are not parallel
Knife not parallel to the block
Impure paraffin
Blunt/dull knife
Block is warm & soft
Knife edge coated w/ paraffin
Thin sections
Microtome screw is loose
Tilt: vertical
Bevel of knife is lost
Incorrect sharpening
Bubble/dirt
Hard spot in the tissue (Ca2+)
Screw/holder is loose
Large & hard blocks
Static electricity
Dirty knife edge
Dull knife edge
Ribbon is split
Nicks/damage on knife
Dirty embedding
Dirty knife
Chatters are seen
Knife vibrates (hard tissue)
Section: sometimes thin &
thick
Blunt knife
Frozen tissue crumbles &
comes off when the block
Knife/block holder is loose
Inadequate freezing
lec.mt 04 |Page | 280
holder when cut
Frozen tissue chips into
fragments
Tissue is frozen too hard
lec.mt 04 |Page | 281
PAS w/ diastase ctrl
Stains
Substance Stained
(+) Color/Result
CHO, Glycogen, Mucins,
PAS (+):Magenta red
Bacteria & Fungi,
basement membrane
Glycogen
Red
Best Carmine
Glycogen
Bright red
Langhan’s iodine method
Glycogen
Mahogany brown
Alcian blue
Acid mucins
Blue
Alcian Blue-PAS
Any mucins
(acid/neutral)
Acid MPS
Sulfated mucins
Carboxylated mucins
Cryptococcus neoformans
Mucins
Acid mucins
Acid mucin: blue
Neutral mucin: magenta
Sulfated mucins: purple
Carboxylated mucins:
blue
Mucin: red
Acid mucins/MPS
Fungi
Stain
PAS
Gomori’s aldehyde fuchsin
stain
Mucicarmine stain
Colloidal (Dialyzed) iron
technique
Acridine orange
Sudan black
Sudan IV (Scharlach R)
Lipids
Lipids (TAG)
Oil red O
Osmium tetroxide
Nile blue sulfate method
Lipids
Lipids
Neutral fat
Cholesterin esters
Cholesterin fatty acids
Fatty acids & soap
Cerebrosides
Brilliant red
Black
= Pinkish red
= Light red
= Light red
= Deep blue to violet
= Light blue
Toluidine blue-acetone mtd
Sulfatide
Borohydride-PeriodicSchiff method
Alkaline fast-green method
Gangliosides
Metachromatic redbrown or yellow
Red
Peracetic acid-Alcian blue
Sakaguchi’s test
Gomori calcium method
Gomori lead method
Lead method
Metal precipitation
Calcium cobalt method
α-naphthyl acetate method
Avoid Ehrlich’s
hematoxylin
Dark blue
Acid MPS: black
Fungi: greenish red
fluorescence
Blue black
red
Histones
Protamines
Cystine
Cysteine
Arginine
Alkaline phosphatase
Acid phosphatase
5’-nucleotidase
ATPase
ATPase
Nonspecific esterase
Comments
Basic fuchsin: essential
component of Schiff
reagent
Method of choice for
glycogen staining
Selective & highly
specific for glycogen
Obsolete
Not specific for glycogen
Avoid celloidinization of
slides
Avoid Ehrlich’s
hematoxylin
Green
Lasts for only 2 hrs
Most commonly used
stain
Nile blue: preliminary
indicator of the type of
lipid present
-Red oxazone (dissol.
neutral lipids)
-Blue oxazone (reacts w/
PL and FFA)
Fast green stains basic
groups in tissues
Blue-green
Orange-red
Brownish-black
Black
Blackish brown deposits
Dark brownish-black ppt
Cobalt phosphate ppt
Reddish brown
Uses Milton reagent
Substrate: β-glyceroPO4
For skel. muscle biopsies
For skel. muscle biopsies
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Stain
Indoxyl acetate method
Tetrazolium method
Feulgen technique
Substance Stained
Nonspecific esterase
Monoamine oxidase
DNA
(+) Color/Result
Blue
Bluish black
Red-purple
Methyl green-pyronin
RNA
DNA
Reticulin fibers
RNA (nucleoli): red
DNA (chromatin): green
black
Collagen
Muscle, cytoplasm, RBC,
fibrin
Collagen & mucus
Muscle, RBC & keratin
Collagen fibers,
cytoplasm, fibroglia
fibrils, axon cylinders,
neuroglia
Elastic fibers
RBCs, myelin sheets
CT
Glomerular basement
membrane
Amyloid & mucous
colloid
Elastic fibers
Elastic fibers
Elastic fibers
Elastic fibers
Fibrin & CT
RBC
RBCs
Muscle
Collagen
Fibrin
Fibrin, muscle striations,
neuroglia, amoeba
RBCs
Myelin
Collagen, osteoid,
cartilage, elastic fibers
Amyloid
Amyloid
= Pink/deep red
= Yellow
Amyloid
Yellow fluorescence
Muscle fibers
Collagen
Muscles, RBC
Collagen
= Red
= Green
= Red
= Yellow
Gomori’s silver
impregnation stain
Van Gieson’s stain
Masson’s trichrome stain
Mallory’s aniline blue
Azocarmine
Weigert’s
Verhoeff’s
Taenzer-Unna-Orcein mtd
Krajian’s technique
Martius-Scarlet-Blue
Mallory’s PTAH
Congo red
Methyl violet-crystal violet
method
Thioflavin-T fluorescent
staining
Modified Gomori’s
Trichrome stain
Lissamine fast red
= Blue
= Red
= Red
Comments
Most reliable & specific
histochemical staining
technique for DNA
Contains Schiff’s reagent
Reticulin = Argyrophilic
(silver stain)
Contains acid fuchsin &
picric acid
(-) Fuchsin: Excellent &
colorful method of
demonstrating CT fibers
= Pale pink/yellow
= Yellow
Heidenhain’s
modification of Mallory’s
aniline blue stain
= Deep blue
Dark-blue/blue-black
Black
Dark-brown
= Bright red
= Dark blue
= Orange-yellow
= Yellow
= Red
= Blue
= Red
= Dark blue
Rapid method
Early fibrin = yellow
Old fibrin = blue
= Blue
= Lighter blue
= Deep brownish-red
Red
Purplish red
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Stain
Schmorl’s Picro-Thionin
method
Substance Stained
Lacunae & canaliculi
Bone matrix
Bielschowsky’s technique
Neurofibril, axons &
dendrites
Neuroglia & collagen
Nerve fibers & nerve
endings
Peripheral neuritis
Axons
Myelin sheath
Neuritic plaques &
tangles
Argentaffin granules
Nissl granules & nucleoli
Nissl granules & nucleoli
Nissl granuls & nucleoli
Nissl substance
Neurons
Myelin sheath
Myelin
Myelin
Astrocytes
Bodian’s stain
Sevier-Munger technique
Toluidine blue
Polychrome methylene blue
Thionine
Cresyl fast violet
Weigert-Pal technique
Luxol fast blue
Weil’s method
Cajal’s gold sublimate
method
Perl’s Prussian blue
Gomori’s Prussian blue
Turnbull’s blue
Benzidine-nitroprusside
stain
Mod. Fouchet’s technique
Gmelin technique
Stein’s iodine test
Schmorl’s ferric
ferricyanide method
Gomori’s aldehyde fuchsin
Mallory’s fuchsin stain
Masson Fontana technique
Von Kossa’s silver nitrate
method
Lindquist modified
rhodanine technique
Hemosiderin
Iron pigments
Ferrous iron
(Hemosiderin)
Hemoglobin & oxidase
granules
Bile pigments
Bile & hematoidin
Bile pigments
(+) Color/Result
= Dark brown-black
= Yellow/brownishyellow
= Black on a grayish BG
Comments
= Lightly stained
Diagnosis of Alzheimer’s
disease
= Black
= Black
= Light brown
= Black
= Black
Deep blue
Deep blue
Purple
= Purple-dark blue
= Pale purple blue
Blue black
Blue-green
Black
Black on a light
brownish BG
Deep blue
Bright blue
Blue
Nissl granules: a.k.a.
Tigroid substances
Dark blue
Emerald to blue green
Blue-purple then green
Depend on the oxidation
of the pigment to green
biliverdin by iodine
Bile, lipofuscins,
melanin, argentaffin
cells, chromaffin, thyroid
colloid
Lipofuscin
Hemofuscin
Melanin
Argentaffin cell granules
Dark blue
Calcium
Black
Copper
Red to orange-red
Purple
Red
= Black
= Black
Argentaffin reaction:
melanin reduces
ammoniacal silver
solutions w/o use of a
reducer
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Stain
Gram-Twort stain
Brown & Brenn method
Wade-Fite technique
Toluidine blue
Substance Stained
Gram (+) organisms
Gram (-) organisms
RBCs
Elastic fibers
Gram (+) bacteria
Gram (-) bacteria
M. leprae
H. pylori
H. pylori
L. pneumophila &
spirochetes
Levaditi’s method
Spirochetes
Modified Steiner & Steiner
Spirochetes, Donovan
technique
bodies, fungi, bacteria
Warthin-Starry method
Spirochetes
Grocott Methenamine Silver Fungi
Cresyl violet acetate mtd
Dieterle method
Lendrum’s phloxinetartrazine method
Orcein method
Giemsa stain
In situ hybridization
PCR
Chondrocalcinosis
Kardasewitsch method
0.1% urea + 5% NaSO4
Metastasis
Degree of localization
Dunn-Thompson
K2MnO4
H2O2
Helly’s
Formalin ammonium
bromide
Alcohol as 1’ fixative
Glutaraldehyde
Carnoy’s
Orth’s
Zenker’s
Formaldehyde
Ethanol
Mucin & glycogen
Mycelia & hyphae
RBCs
Viral inclusions
(+) Color/Result
= Blue-black
= Pink-red
= Green
= Black
= Blue
= Red
Golden yellow
Dark blue against blue
BG
Blue-violet
Dark brown to black
Comments
Black on a yellowish BG
Black
Black
= Sharply outlined I
black
= Gray-black
= Old rose
= Yellow
Bright red
HBsAg
Brown-black
Bacteria
= Blue
Recommended for blood
Mast cell granules
= Deep blue
and BM parasites,
Eosinophilic granules
= Red
inclusion conjunctivitis,
Nuclei
= Blue
Toxoplasma, spirochetes
Cytoplasm
= Pink
& other bacteria
Most sensitive technique for identifying DNA
DNA amplification
Pseudogout
Pigment removal
70% ETOH + 28% NH3 water
Remove yellow color of HNO3
Most definitive of malignancy
Most reliable indicator of prognosis of malignant tumors
Hgb = emerald green
Removes excess melanin
Contains formalin, K2CrO4 and HAc
Fixative for CNS (gold/silver stain)
Increased tissue shrinkage
Not satisfactory for PAS
Nonaqueous fixative
Pheochromocytoma
PTAH for cross-striations
Wash tissue in water after fixation in Zenker’s
Combines w/ amino group
Nonadditive fixative
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pH >6.0 (formalin)
Universal solvents
Soft paraffin
Weigert’s hematoxylin
Natural resins (mounting)
Formalin-alcohol
Churukian-Schenk
technique
Masson-Fontana
Muscle biopsies
Paraffin sections
Zamboni’s PAF
Glutaraldehyde
10% NBF
Paraformaldehyde
Warthin-Starry stain
Iris diaphragm
Substage condenser
Pathology
Cornelius Celsus
Littoral cells
Hoffbauer cells
Cancer
Biohazards
Mercuric chloride
According to the presence
of a mordant
According to the presence
of differentiator
According to the resultant
color
Vital staining
Neurons
Prevent brown microcrystalline deposits in H & E stain
Both dehydrates & clears
For thick sections
Not easily decolorized w/ acidic solutions
Inherently acidic
Microincineration
Substance that can bind silver but need a chemical reducer
Substance that can both bind & reduce silver
Isopentane at -150’C
If isopentane is low, dust muscle w/ talc then freeze in liquid nitrogen
Naphthol AS-D chloroacetate esterase
Specimens may remain indefinitely
Specimens may be removed after 2-4 hrs
pH 7.2-7.4
Pure polymer of formaldehyde
Calibrate pH meter to 7.0
Increase amount of light
Adjust to focus the image of the substage diaphragm
Greek: Pathos = suffering
1st to describe the 4 signs of inflammation
Splenic macrophages
Placental macrophages
Latin: Cancrum = crab
Infectious agents
Contaminated solutions
Contaminated specimens
Corrode all metals except for the nickel alloy Monel
Methods of Staining
a. Direct staining = w/o mordant
b. Indirect staining = w/ mordant: serves as a link/bridge between the tissue &
the dye
a. Progressive = w/o differentiator/decolorizer
b. Regressive = w/ differentiator/decolorizer
*1’ stain = acidic (decolorizer: basic)
*2’ stain = basic (decolorizer: acidic)
a. Orthochromatic = “ortho”: correct/same | same color = dye & tissue
b. Metachromatic = “meta”: after/change | different color = dye & tissue
Selective staining of living cells
a. Intravital stain = injection of dye  animal body
- Ex. Lithium, Carmine, India ink
b. Supravital stain = staining of cells immediately after removal from the animal
body. Examples are:
- Neutral red = Best vital dye
- Janus green = mitochondria
- Trypan blue
- Nile blue
- Thionine
- Toluidine blue
Functional cells of the CNS
Nerve fibers:
a. Dendrites (Greek: “Tree”) = conduct impulses to the cell body
lec.mt 04 |Page | 286
b. Axons = conduct impulses away from the cell body
Criteria for the diagnosis of Marked progesterone effect
normal pregnancy
At least 50% of intermediate cells in clusters
At least some typical pregnancy cells present
<30% of matured superficial cells
Doderlein-filled dirty BG
Staining solutions used in
a. Hematoxylin = dark purple to black
Pap’s staining method
b. OG-6 = orange w/ a hint of green
(Macroscopic)
c. EA 36/50 = olive green w/ a hint of brown & red
EA 36/50
Components:
a. Eosin Y
b. Bismarck brown
c. Light green SF
# in EA designates the proportion of SF
♫ Affinity:
OG-6
Matured superficial cells
Keratinizing malignant cells
♫ Cytoplasmic:
Bright orange to yellow orange
♫ Affinity:
EA 36/50
Immature vaginal cells (parabasal, intermediate)
♫ Cytoplasm color:
Transparent blue to gray to brown hue
Presently, the Bethesda system divides squamous cell abnormalities into 4 categories:
ASCUS
Atypical cells of undetermined significance
L-SIL
Low-grade squamous intraepithelial lesion
H-SIL
High-grade squamous intraepithelial lesion
SCC
Squamous cell carcinoma
Description
Bethesda 2001
Papanicolau
Normal
(-) for intraepithelial lesion/malignancy
Class I
Atypical
ASCUS
Class II
HPV
L-SIL
Class II
Mild dysplasia
L-SIL
Class II
Moderate dysplasia
H-SIL
Class III
Severe dysplasia
H-SIL
Class III
Carcinoma in situ
H-SIL
Class IV
Invasive carcinoma
Invasive carcinoma
Class V
Microtome
Usual
Description
Tissues embedding in
Microtome
Knives
Length
Plane concave
25 mm One side flat, other Less concave: celloidin-impregnated tissues Sliding
is concave
More concave: paraffin embedded tissues
Biconcave
120 mm
Plane wedge
100 mm
Carmine
Best Carmine
Mucicarmine
Both sides are
Paraffin
concave
Both sides are
Frozen sections
straight
Hard, tough tissue specimen (paraffin)
Chromatin stain
Carmine + Aluminum chloride = For glycogen
Carmine + Aluminum hydroxide = For C. neoformans and mucin
Base-sledge
Rotary
Rocking
Rotary
Sliding
Base-sledge
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Picrocarmine
Duke’s staging for neoplasia
of the rectum
Biopsy
Types of Biopsy
Exfoliative cytology
Excisional biopsy
Incisional biopsy
Needle biopsy
Bite biopsy
Cutaneous biopsy
Punch biopsy
Shave biopsy
Wedge biopsy
Marginal excision
Carmine + Picric acid = for neuropathological studies
One of the most frequently applied for staging individual tumors
Biopsy
Excision and exam (living subject)
Preferred: perform the biopsy at the periphery of the tumor (advancing tumor
margin)
Desquamated cells
Sex hormonal status in females
Sex chromatin phenotype
Complete removal of a lesion
Most reliable
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
Aspiration of fluid
Small pcs of tumor are removed w/ special forceps
Skin fragments
For specimens >2mm  embed in a single paraffin block
Curettage specimens
Specimen is subdivided w/ a razor blade
Shell-out end
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