MUST TO KNOW IN HISTOPATHOLOGIC TECHNIQUES

Germ layers 1. Ectoderm

2. Mesoderm
3. Endoderm
Categories of tissues 1. Epithelial = 3 germ layers
2. Nervous = endoderm
3. Muscular = mesoderm
4. Connective = mesoderm
Covering Epithelia
Simple squamous Bowman’s, endothelium, loop of Henle, alveoli
Simple cuboidal Ducts of glands, walls of thyroid follicles
Simple columnar Gallbladder (nonciliated)
Uterine tube (ciliated)
Stratified squamous Skin (keratinized)
Vagina, esophagus, cervix (nonkeratinized)
Stratified columnar Male urethra
Pseudostratified columnar Female reproductive tract (nonciliated)
Trachea (ciliated), Epididymis
Glandular Epithelia
Exocrine glands w/ ducts
Tubular = stomach, uterus
Acinar/alveolar = pancreas, salivary glands
Tubuloacinar = prostate
Endocrine glands Ductless
Pancreas Exocrine = enzymes
Endocrine = hormones
Merocrine No loss of cytoplasm
Goblet cells, sweat glands
Apocrine w/ cytoplasmic loss
Distal portion is pinched off
Mammary glands
Holocrine Disintegrating cell and its constituents released
Complete breakdown of cell
Sebaceous gland
Connective Tissues
Connective tissue Support
Framework
Cells are widely separated
Collagen 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

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3. Tumor Swelling
Fluid extravasation
4. Dolor Pain
Sensory nerves
5. Functio laesa 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

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 Reversible
One type of adult cell ↔ Changes in structural components
Anaplasia/ Irreversible
Dedifferentiation Criterion toward malignancy
Adult cell More primitive cells (release tumor markers)
Neoplasia/tumor Continuous abnormal proliferation of cells w/o control (no purpose/function)
Ex. Leukemia
Oncology Study of neoplasm
Tumors
Parts of a tumor 1. Parenchyma = active elements (tumor cells)
2. Stroma = CT framework
Types of tumor 1. Capacity to produce death:
- Benign (Ex. mole)
- Malignant
2. Histologic characteristics:
- Medullary = cells (parenchyma) > supporting tissues (stroma)
- Scirrhous = supporting tissues (stroma) > cells (parenchyma)
Benign “-oma”
Malignant “SaMe CarE”
“-sarcoma” = mesenchymal/CT
“-carcinoma” = epithelial tissues
Leukemia Malignant
Lymphoma
Squamous cell papilloma Benign
Squamous cell carcinoma Malignant
Hepatoma/ Malignant
hepatocarcinoma
Melanoma/ Malignant
melanocarcinoma
Ectopic pregnancy Fallopian tube pregnancy
Grading
Grading Aggressiveness/level of malignancy
Differentiated cells = resemble normal cells
Undifferentiated cells = younger cells
Broder’s Classification (Grading)
Grade Differentiated Cells Undifferentiated Cells Treatment
I 75-100% 0-25% Surgery
II 50-75% 25-50% ↓
III 25-50% 50-75% ↓
IV 0-25% 75-100% Radiation
Staging
Staging Size, extent of spread to lymph nodes, +/- metastases
UICC TNM classification
AJCS Grading + staging
TNM System
TNM system Applicable to all forms of neoplasia
T 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
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 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)
N 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
M Metastasis
M0 = no evidence of metastases
M1 = distant metastases are present
Mx = distant metastases not evaluable
Teratomas Compound tumors
Greek: Monstrous tumors
May contain hair, teeth, bones
w/ heartbeat
Cellular Death
Apoptosis Programmed cell death (cellular suicide)
Necrobiosis Physiologic cell death
Ex. normal sloughing off of skin cells
Necrosis Pathologic cell death
Types of Necrosis
Coagulation necrosis Most common
Tombstone formation
“MyLKS”
Myocardium
Lungs
Kidneys
Spleen
Liquefaction/colliquative Pus formation
necrosis Brain & spinal cord
Caseous/caseation necrosis Yellow, cheesy, crumbly material
TB, syphilis, tularemia, lymphogranuloma inguinale
Gangrenous necrosis Sulfide gas production
a. Dry gangrene = arterial occlusion
b. Wet gangrene = venous occlusion
Fat necrosis Chalky white precipitates
Pancreatic degeneration
Fatty degeneration Liver
Somatic death
1’ changes During somatic death
“CRC”: circulatory, respiratory, CNS failure
2’ changes After somatic death
“ARLP DPA”: Algor mortis, Rigor mortis, Livor mortis, Postmortem clotting,
Dessication, Putrefaction, Autolysis
Algor mortis (1st) Postmortem cooling

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 Cooling: 7’F/hr
Rigor mortis (2nd) 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
Fixation ♫ 50% alcohol = All types
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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 c. touch preparation/impression
d. pull-apart
Never touch the bottom of the fixative container
Fixing smear Equal parts of ethanol & ether = BEST (but highly flammable)
95% ethanol = commonly used
Spray fixatives = 1 ft away
Sputum Saccomanno’s fixative
3 specimen
(+) alveolar macrophage = sputum
(-) alveolar macrophage = saliva
BAL P. carinii/P. jiroveci
Jelly-like clots Prevent by adding 300U heparin/100mL aspirate
GI specimen If >½ hr delay of fixation digestion of cells
Fasting: 8 hrs
Urine 50mL = cytology
10-15mL = UA
2nd urine = preferred
Pap smear 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)
Adhesive agents Pooled human serum/plasma
Celloidin ether alcohol
Leuconostoc culture
3 primary materials used 1. Speculum
for obtaining specimen for 2. Ayer’s spatula = rotate 3600
Pap smear 3. Cytobrush = Os
Strawberry cervix T. vaginalis
Cells (Cervicovaginal Smears)
Parabasal | Intermediate | Superficial
↓ ----------Estrogen----------↑
Shift to the left
Shift to the right
Shift to the midzone
Superficial cells 45-50μm
Pyknotic nucleus
True acidophilia

Intermediate cells 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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Doderlein bacillus L. acidophilus
Pap’s stain: blue to lavender
C. albicans Diabetic patients
Sish kebab appearance
T. vaginalis Pear-shaped, blue-gray to blue-green
Pigs on a scruff appearance
Leptothrix Indicates T. vaginalis infection
G. vaginalis Clue cells
Koilocytes Wrinkled prune appearance
w/ perinuclear halo
HPV (LSIL)
Ferning Formation of salt crystals

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/ estrogen MI = 0/20/80
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 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 48 hours
Blood smears (routine) 7 days
Microbiology smears 7 days
BB donor/recipient 7 days post-transfusion
specimens
Pathology/BM slides 10 years
Pathology blocks 10 years
Cytogenetic slides 3 years
Cytogenetics diagnostic 20 years
images
Forensic Cases
Body fluids 1 year
Tissue for toxicology 1 year
Wet tissues 3 years
Paraffin blocks Indefinitely
Slides Indefinitely
Reports Indefinitely
Gross photographs/ Indefinitely
negatives
Dried blood films Indefinitely
Frozen tissue for DNA indefinitely
Autopsy (Postmortem Examination)
Autopsy 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
Types of autopsy 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)
Preliminaries for PME 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 Burial & cremation purposes

Certificate Transport of body from hospital funeral cemetery
Medical insurance claiming
- If suicide: (-) insurance
- Acts of God (lightning, flood): (-) insurance
- Civil war: (-) insurance
PME is permitted w/o 1. When it is ordered by the police or coroner (NBI)
consent in the following 2. When it is necessary to complete the death certificate
circumstances 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
If pathologist is not The medico-legal examiner or the coroner has jurisdiction in medico-legal cases
available & may authorize the pathologist to proceed w/ an autopsy
The coroner has authority 1. All natural deaths occurring in the hospital w/in 24 hrs of admission, unless
in the following cases 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
Somatic death Death of an organism
Cessation of circulation & respiration (1960’s)
Criteria for the 1. Advanced resuscitation techniques that are capable of reviving effectively
pronouncement of death 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 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
American bar association & 1. irreversible cessation of circulation & respiratory functions
national conference of 2. Irreversible cessation of all functions of the entire brain, including the
commission of uniform brainstem is dead
state laws legislative
definition of death (1980)
American academy of Death:
neurology 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
Postmortem changes 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
Technique of Virchow Organs removed & dissected individually in the body
Most widely used metohd
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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
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Cryostat procedure Temperature: -18 to -20’C
(Cold microtome) Cryostat: refrigerated cabinet w/ rotary microtome
O.C.T. (Optimal Cutting Best mounting media for cryostat sections
Temperature)

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 1 and most critical step
st

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
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 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
Flemming’s w/ acetic acid
Newcomer’s
Carnoy’s
Heidenhain’s SuSa
Cytoplasmic fixatives “HORFF”
Helly’s
Orth’s
Regaud’s

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 Flemming’s w/o acetic acid
Formalin w/ post chroming
Histochemical fixatives “FANA”
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 immunocytochemistry
paraformaldehyde-
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
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 fixatives “ROCK”
a. Regaud’s (Moller’s) = chromatin, mitochondria, mitotic figures…
b. Orth’s = for Rickettsia, tissue necrosis

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 c. Chromic acid = preserves CHO
d. K2CrO4 = mitochondria (if acidified, fixes chromatin bodies & chromosomes
but destroys mitochondria)
Chromate pigments Fine, yellow brown
Lead fixatives Used in 4% aqueous solution of basic lead acetate
For acid MPS and mucin
Picric acid fixatives 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)
Glacial acetic acid Solidifies at 17’C
Fixes & precipitates nucleoproteins, chromosomes, & chromatin material
Most commonly combined w/ other fixatives
Alcoholic 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
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 acid Precipitates proteins
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-chromatization Primarily fixed tissue 2.5-3% K2CrO4 (mordant)
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”

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
(+) Fat Fatty tissues: cut in thin sections, fixed longer
(+) Blood Flush out w/ NSS fix
Cold temperature Inactivates enzymes
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 Color Removed by:

Acid formaldehyde hematin Brown/black granules “SAKaL”
a. Saturated picric acid
b. Alcoholic KOH
c. Kardasewitsch method
d. Lillie’s method
Mercuric chloride pigment Black granules Alcoholic iodine
Chromate pigment Fine, yellow brown Acid-alcohol
Osmium tetroxide pigment Black precipitate crystals Cold H2O
Crush artifact Intense eosinophilic staining at the center of the tissue (H & E)
Due to partial coagulation of partially fixed protein
Decalcification
20:1 Ratio of decalcifying agent to tissue
37’C Impaired nuclear stain by Van Gieson’s stain
55’C Tissue Digestion (24-48 hrs)
RT (18-30’C) Optimum temperature
24-48 hrs Time
Decalcifying agents Acids
Chelating agents (EDTA/versene)
Ion exchange resins
Elec. ionization (electrophoresis)
HNO3 Most common
a. Perenyi’s = tissue softener & decalcifying agent
b. Phloroglucin-HNO3 = most rapid
- Disadvantage: Yellow color on tissue (neutralize w/ sodium thiosulfate)
5% Formic acid Both fixative & decalcifying agent
Best general decalcifying agent
For small pcs of bones & teeth
HCl (Von Ebner’s) For small pcs of bones & teeth
For surface decalcification (HCl)
EDTA For EM, IHC, & enzyme staining
Ion exchange resins Hastens decalcification by removing calcium ions from formic acid-containing
decalcifying solutions
Electrophoresis Ca2+ are attracted to negative electrode (cathode)
Measuring extent of Physical method
decalcification 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
Post-Decalcification Removal/neutralization of acid from the tissues after decalcification
Lithium carbonate or sodium bicarbonate solution
Tissue softeners 4% phenol
Molliflex = tissues appear swollen & soapy
2% HCl
lec.mt 04 |Page | 271
 1% HCl in 70% alcohol
Dehydration
Dehydration Aim: To remove fixative & H2O
Ascending grades of alcohol (Start: 65%)
Embryonic & animal tissues: 30% ETOH
10:1 Ratio of dehydrating agent to tissue
Ethanol Best dehydrating agent
Methanol Blood & tissue films
Butanol Plants & animals
Denatured alcohol Ethanol + methanol
Acetone Both fixative & dehydrating agent
Dioxane Both dehydrating & clearing agents
(Diethylene dioxide)
Tetrahydrofuran (THF)
Graupner’s method Dehydration w/ dioxane
Weiseberger’s method
Cellosolve Ethylene glycol monoethyl ether
Combustible and toxic
Triethyl phosphate --
Additives to dehydrating a. 4% phenol + 95% ETOH = softener
agents b. Anhydrous CuSO4 (Last ETOH bath)
- both dehydrating agent & indicator of H2O content of 100% ETOH
- (+) H2O = White Blue
Methods of determining 1. Anhydrous CuSO4 method
incomplete dehydration 2. Xylene Milky
Clearing
Xylene (Xylol) Most commonly used
Clearing time: ½ to 1 hr
Block size: <5mm
Toluene Substitute for xylene/benzene
Clearing time: 1-2 hrs
Not carcinogenic
Toxic fumes
Chloroform Toxic to liver
For clearing tough tissues
Does not make tissue translucent but removes alcohol
Benzene For urgent biopsies
Minimum shrinkage
Aplastic anemia
Methyl salicylate For double embedding techniques
Methyl benzoate
Cedarwood oil For CNS, smooth muscles, skin
Clove oil Minimum shrinkage
Has tendency to become adulterated
CCl4 Similar to chloroform but is cheaper
Disadvantage: similar to chloroform
Aniline oil For delicate tissues, embryos and insects
Glycerin No dealcoholization but make the tissues clearer
Gum syrup
Others Citrus fruits oil
Trichloroethane & petrol
Impregnation
lec.mt 04 |Page | 272
25:1 Ratio of infiltrating medium to tissue
Medium Paraffin wax
Celloidin (collodion)
Gelatin = H2O soluble, not a wax
Plastic = EM
Paraffin 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
Paraffin filters Filtration at 2’C above the MP of wax
Ex. Green’s no. 904 (coarse filter paper)
Paraffin oven 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 For unfixed, unfrozen tissues
For enzyme demonstration
Ultrathin microtome EM
Diamond knives or broken plate glass
Freezing microtome Queckett
Clearance angle Knife to tissue block: 0-150 angle
Bevel angle 27-320 angle

Honing Removal of gross nicks

Heel to toe (Edge 1st)
Types of hones a. Belgium yellow: Best
b. Arkansas
c. Fine carborundum: for badly nicked knives
Stropping 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
Natural dyes From plants & animals
a. Hematoxylin
b. Cochineal dyes: female Coccus cacti
c. Orcein: from lichens
d. Saffron
Synthetic dyes A.k.a. coal tar dyes
Derived from benzene & collectively known as aniline dyes
Chromophores Greek: “color-bearer”
Coloring property
Auxochrome Greek: “increasers”
Dyeing property
Chromogen Benzene + Chromophore
Imparts color temporarily
Dye Chromogen + auxochrome
Imparts color to tissue almost permanently
Chromophores a. Quinoid ring: Basic fuchsin
b. Azo groups: Congo red
c. Xanthene: Eosin
d. Quinone-imine group
- Oxazin: cresyl fast violet
- Thiazins: toluidine blue
Auxochromes Cationic auxochromes: amino group (NH3+)
Anionic auxochromes: hydroxyl (OH-) and carboxyl (COO-) groups
Dye modifiers Attached on benzene ring
a. Ethyl group
b. Methyl group
c. Sulphonic acid
Van der Waals forces Alum hematoxylin
Sudanophilia Tissue stained w/ fat or oil-soluble dyes
H & E Staining
Hematoxylin 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
H 2O 2
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 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
Benzidine Hgb
Acridine orange RNA (red fluorescence)
DNA (green fluorescence)
Gentian violet Crystal violet + methyl violet + dextrin
Congo red Elastic tissue, amyloid, myelin
Iodine Oldest stain
Malachite green Ascaris eggs
Janus green Mitochondria (intravital stain)
Night blue Substitute for carbolfuchsin
Victoria blue Neuroglia
Lysochromes Oil soluble dyes
a. SBB = Black (most sensitive)
b. Sudan III = orange
c. Sudan IV (Scharlach R) = red
Adhesives 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)
Mounting media ♫ 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
Stains on skin Remove by using 0.5% acid alcohol tap water
Restaining of old sections 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
Broken slides 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

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
Skeletal muscle

lec.mt 04 |Page | 278

 Cardiac muscle
Vimentin Melanomas
Schwannomas
Desmin Leiomyoma (smooth muscle)
Rhabdomyosarcoma (skeletal muscle)
GFAP (Glial Fibrillary Acidic Astrocytoma
Protein)
NF (Neurofilament) Neuroblastoma
Ganglioneuromas
Neuroma
Chemodectoma
Pheochromocytoma
S100 protein Low MW Ca2+-binding protein
CNS glial cells, Schwann cells
Neuroendocrine Markers
NSE (Neuron-specific Strong evidence of neural/neuroendocrine differentiation
enolase)
Others 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
Ribbon is split
Chatters are seen
Section: sometimes thin &
thick
Frozen tissue crumbles &
comes off when the block
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
Nicks/damage on knife
Dirty embedding
Dirty knife
Knife vibrates (hard tissue)
Blunt knife
Knife/block holder is loose
Inadequate freezing
lec.mt 04 |Page | 280
holder when cut
Frozen tissue chips into Tissue is frozen too hard
fragments

lec.mt 04 |Page | 281

 Stains
Stain Substance Stained (+) Color/Result Comments
PAS CHO, Glycogen, Mucins, PAS (+):Magenta red Basic fuchsin: essential
Bacteria & Fungi, component of Schiff
basement membrane reagent
PAS w/ diastase ctrl Glycogen Red Method of choice for
glycogen staining
Best Carmine Glycogen Bright red Selective & highly
specific for glycogen
Langhan’s iodine method Glycogen Mahogany brown Obsolete
Not specific for glycogen
Alcian blue Acid mucins Blue Avoid celloidinization of
slides
Alcian Blue-PAS Any mucins Acid mucin: blue Avoid Ehrlich’s
(acid/neutral) Neutral mucin: magenta hematoxylin
Gomori’s aldehyde fuchsin Acid MPS Sulfated mucins: purple
stain Sulfated mucins Carboxylated mucins:
Carboxylated mucins blue
Mucicarmine stain Cryptococcus neoformans Mucin: red Avoid Ehrlich’s
Mucins hematoxylin
Colloidal (Dialyzed) iron Acid mucins Dark blue
technique
Acridine orange Acid mucins/MPS Acid MPS: black Lasts for only 2 hrs
Fungi Fungi: greenish red
fluorescence
Sudan black Lipids Blue black
Sudan IV (Scharlach R) Lipids (TAG) red Most commonly used
stain
Oil red O Lipids Brilliant red
Osmium tetroxide Lipids Black
Nile blue sulfate method Neutral fat = Pinkish red Nile blue: preliminary
Cholesterin esters = Light red indicator of the type of
Cholesterin fatty acids = Light red lipid present
Fatty acids & soap = Deep blue to violet -Red oxazone (dissol.
Cerebrosides = Light blue neutral lipids)
-Blue oxazone (reacts w/
PL and FFA)
Toluidine blue-acetone mtd Sulfatide Metachromatic red-
brown or yellow
Borohydride-Periodic- Gangliosides Red
Schiff method
Alkaline fast-green method Histones Green Fast green stains basic
Protamines groups in tissues
Peracetic acid-Alcian blue Cystine Blue-green
Cysteine
Sakaguchi’s test Arginine Orange-red Uses Milton reagent
Gomori calcium method Alkaline phosphatase Brownish-black
Gomori lead method Acid phosphatase Black Substrate: β-glyceroPO4
Lead method 5’-nucleotidase Blackish brown deposits
Metal precipitation ATPase Dark brownish-black ppt For skel. muscle biopsies
Calcium cobalt method ATPase Cobalt phosphate ppt For skel. muscle biopsies
α-naphthyl acetate method Nonspecific esterase Reddish brown
lec.mt 04 |Page | 282
 Stain Substance Stained (+) Color/Result Comments
Indoxyl acetate method Nonspecific esterase Blue
Tetrazolium method Monoamine oxidase Bluish black
Feulgen technique DNA Red-purple Most reliable & specific
histochemical staining
technique for DNA
Contains Schiff’s reagent
Methyl green-pyronin RNA RNA (nucleoli): red
DNA DNA (chromatin): green
Gomori’s silver Reticulin fibers black Reticulin = Argyrophilic
impregnation stain (silver stain)
Van Gieson’s stain Collagen = Pink/deep red Contains acid fuchsin &
Muscle, cytoplasm, RBC, = Yellow picric acid
fibrin
Masson’s trichrome stain Collagen & mucus = Blue
Muscle, RBC & keratin = Red
Mallory’s aniline blue Collagen fibers, = Red (-) Fuchsin: Excellent &
cytoplasm, fibroglia colorful method of
fibrils, axon cylinders, demonstrating CT fibers
neuroglia
Elastic fibers = Pale pink/yellow
RBCs, myelin sheets = Yellow
Azocarmine CT Heidenhain’s
Glomerular basement modification of Mallory’s
membrane aniline blue stain
Amyloid & mucous = Deep blue
colloid
Weigert’s Elastic fibers Dark-blue/blue-black
Verhoeff’s Elastic fibers Black
Taenzer-Unna-Orcein mtd Elastic fibers Dark-brown
Krajian’s technique Elastic fibers = Bright red Rapid method
Fibrin & CT = Dark blue
RBC = Orange-yellow
Martius-Scarlet-Blue RBCs = Yellow Early fibrin = yellow
Muscle = Red Old fibrin = blue
Collagen = Blue
Fibrin = Red
Mallory’s PTAH Fibrin, muscle striations, = Dark blue
neuroglia, amoeba
RBCs = Blue
Myelin = Lighter blue
Collagen, osteoid, = Deep brownish-red
cartilage, elastic fibers
Congo red Amyloid Red
Methyl violet-crystal violet Amyloid Purplish red
method
Thioflavin-T fluorescent Amyloid Yellow fluorescence
staining
Modified Gomori’s Muscle fibers = Red
Trichrome stain Collagen = Green
Lissamine fast red Muscles, RBC = Red
Collagen = Yellow
lec.mt 04 |Page | 283
 Stain Substance Stained (+) Color/Result Comments
Schmorl’s Picro-Thionin Lacunae & canaliculi = Dark brown-black
method Bone matrix = Yellow/brownish-
yellow
Bielschowsky’s technique Neurofibril, axons & = Black on a grayish BG
dendrites
Neuroglia & collagen = Lightly stained
Bodian’s stain Nerve fibers & nerve Diagnosis of Alzheimer’s
endings disease
Sevier-Munger technique Peripheral neuritis = Black
Axons = Black
Myelin sheath = Light brown
Neuritic plaques & = Black
tangles
Argentaffin granules = Black
Toluidine blue Nissl granules & nucleoli Deep blue
Polychrome methylene blue Nissl granules & nucleoli Deep blue
Thionine Nissl granuls & nucleoli Purple
Cresyl fast violet Nissl substance = Purple-dark blue Nissl granules: a.k.a.
Neurons = Pale purple blue Tigroid substances
Weigert-Pal technique Myelin sheath Blue black
Luxol fast blue Myelin Blue-green
Weil’s method Myelin Black
Cajal’s gold sublimate Astrocytes Black on a light
method brownish BG
Perl’s Prussian blue Hemosiderin Deep blue
Gomori’s Prussian blue Iron pigments Bright blue
Turnbull’s blue Ferrous iron Blue
(Hemosiderin)
Benzidine-nitroprusside Hemoglobin & oxidase Dark blue
stain granules
Mod. Fouchet’s technique Bile pigments Emerald to blue green
Gmelin technique Bile & hematoidin Blue-purple then green
Stein’s iodine test Bile pigments Depend on the oxidation
of the pigment to green
biliverdin by iodine
Schmorl’s ferric Bile, lipofuscins, Dark blue
ferricyanide method melanin, argentaffin
cells, chromaffin, thyroid
colloid
Gomori’s aldehyde fuchsin Lipofuscin Purple
Mallory’s fuchsin stain Hemofuscin Red
Masson Fontana technique Melanin = Black Argentaffin reaction:
Argentaffin cell granules = Black melanin reduces
ammoniacal silver
solutions w/o use of a
reducer
Von Kossa’s silver nitrate Calcium Black
method
Lindquist modified Copper Red to orange-red
rhodanine technique

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 Stain Substance Stained (+) Color/Result Comments
Gram-Twort stain Gram (+) organisms = Blue-black
Gram (-) organisms = Pink-red
RBCs = Green
Elastic fibers = Black
Brown & Brenn method Gram (+) bacteria = Blue
Gram (-) bacteria = Red
Wade-Fite technique M. leprae Golden yellow
Toluidine blue H. pylori Dark blue against blue
BG
Cresyl violet acetate mtd H. pylori Blue-violet
Dieterle method L. pneumophila & Dark brown to black
spirochetes
Levaditi’s method Spirochetes Black on a yellowish BG
Modified Steiner & Steiner Spirochetes, Donovan Black
technique bodies, fungi, bacteria
Warthin-Starry method Spirochetes Black
Grocott Methenamine Silver Fungi = Sharply outlined I
black
Mucin & glycogen = Gray-black
Mycelia & hyphae = Old rose
RBCs = Yellow
Lendrum’s phloxine- Viral inclusions Bright red
tartrazine method
Orcein method HBsAg Brown-black
Giemsa stain 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
In situ hybridization Most sensitive technique for identifying DNA
PCR DNA amplification
Chondrocalcinosis Pseudogout
Kardasewitsch method Pigment removal
70% ETOH + 28% NH3 water
0.1% urea + 5% NaSO4 Remove yellow color of HNO3
Metastasis Most definitive of malignancy
Degree of localization Most reliable indicator of prognosis of malignant tumors
Dunn-Thompson Hgb = emerald green
K2MnO4 Removes excess melanin
H2 O 2
Helly’s Contains formalin, K2CrO4 and HAc
Formalin ammonium Fixative for CNS (gold/silver stain)
bromide
Alcohol as 1’ fixative Increased tissue shrinkage
Glutaraldehyde Not satisfactory for PAS
Carnoy’s Nonaqueous fixative
Orth’s Pheochromocytoma
Zenker’s PTAH for cross-striations
Wash tissue in water after fixation in Zenker’s
Formaldehyde Combines w/ amino group
Ethanol Nonadditive fixative
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pH >6.0 (formalin) Prevent brown microcrystalline deposits in H & E stain
Universal solvents Both dehydrates & clears
Soft paraffin For thick sections
Weigert’s hematoxylin Not easily decolorized w/ acidic solutions
Natural resins (mounting) Inherently acidic
Formalin-alcohol Microincineration
Churukian-Schenk Substance that can bind silver but need a chemical reducer
technique
Masson-Fontana Substance that can both bind & reduce silver
Muscle biopsies Isopentane at -150’C
If isopentane is low, dust muscle w/ talc then freeze in liquid nitrogen
Paraffin sections Naphthol AS-D chloroacetate esterase
Zamboni’s PAF Specimens may remain indefinitely
Glutaraldehyde Specimens may be removed after 2-4 hrs
10% NBF pH 7.2-7.4
Paraformaldehyde Pure polymer of formaldehyde
Warthin-Starry stain Calibrate pH meter to 7.0
Iris diaphragm Increase amount of light
Substage condenser Adjust to focus the image of the substage diaphragm
Pathology Greek: Pathos = suffering
Cornelius Celsus 1st to describe the 4 signs of inflammation
Littoral cells Splenic macrophages
Hoffbauer cells Placental macrophages
Cancer Latin: Cancrum = crab
Biohazards Infectious agents
Contaminated solutions
Contaminated specimens
Mercuric chloride Corrode all metals except for the nickel alloy Monel
Methods of Staining
According to the presence a. Direct staining = w/o mordant
of a mordant b. Indirect staining = w/ mordant: serves as a link/bridge between the tissue &
the dye
According to the presence a. Progressive = w/o differentiator/decolorizer
of differentiator b. Regressive = w/ differentiator/decolorizer
*1’ stain = acidic (decolorizer: basic)
*2’ stain = basic (decolorizer: acidic)
According to the resultant a. Orthochromatic = “ortho”: correct/same | same color = dye & tissue
color b. Metachromatic = “meta”: after/change | different color = dye & tissue
Vital staining 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
Neurons Functional cells of the CNS
Nerve fibers:
a. Dendrites (Greek: “Tree”) = conduct impulses to the cell body
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 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
OG-6 ♫ Affinity:
Matured superficial cells
Keratinizing malignant cells
♫ Cytoplasmic:
Bright orange to yellow orange
EA 36/50 ♫ Affinity:
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

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
Carmine Chromatin stain
Best Carmine Carmine + Aluminum chloride = For glycogen
Mucicarmine Carmine + Aluminum hydroxide = For C. neoformans and mucin
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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

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