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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 Female reproductive tract (nonciliated)
columnar 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)

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BM (reticular)
Lymph node (reticular)
Embryo (mesenchyme)
Hypodermis

Dense CT Dermis
Capsules
Tendons
Stroma of cornea
Special Connective Tissues
Cartilage Hyaline = trachea
Fibrous = intervertebral discs
Elastic = ear, epiglottis
Bone Cancellous/spongy/trabecular = epiphysis/ends of long bones
Compact/cortical = diaphysis/shaft
Others Blood
Lymph
Hematopoietic tissues
Acid Fixative: Lead fixatives
mucopolysaccharides Stain: Alcian blue
Osteogenesis imperfect Brittle bone disease
Defective production of collagen
Deposits found in Connective Tissues (Eosinophilic)
Fibrin Early: yellow
Old: blue
Stains:
Mallory‟s PTAH
Lendrum‟s MSB
Fibrinoid Necrotizing vasculitis
Staining reactions identical to fibrin
Mixture of exudates & altered cytoplasmic constituents
Hyaline Degenerated collagen
Hypertension, atheroma, diabetic kidney
Stain: PAS
Amyloid TB, leprosy, osteomyelitis
Stains: “CoMT”
Congo Red
Metachromatic stain
Thioflavine
Muscle Tissues
Smooth Involuntary
Intestines, blood vessels
Skeletal Striated, voluntary
Skeletal muscles
Cardiac Striated, involuntary
Heart
Nervous Tissues

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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

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 Intergrade between acute & chronic
inflammation
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 Blood + exudates
inflammation Bacterial infections & other infections
Suppurative/purulent
inflammation debris

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

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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 Ex. Enlargement of one kidney
hyperplasia
Pathologic hyperplasia Ex. Endometrial hyperplasia
Congenital hypertrophy Phenytoin-induced
Degenerative Changes = Tissues have abnormalities
Metaplasia Reversible
One adult cell type ↔ Another adult cell type
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

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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 Undifferentiated Treatment
Cells Cells
I 75-100% 0-25% Surgery
II 50-75% 25-50% ↓
III 25-50% 50-75% ↓
Radiation
IV 0-25% 75-100%
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
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

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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 Yellow, cheesy, crumbly material
necrosis 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
st
Algor mortis (1 ) Postmortem cooling
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 Opposite of postmortem lividity
pressure is released)
Oozing of blood (incision) No oozing of blood (incision)
Postmortem Clot vs. Antemortem Clot

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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
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)

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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 1. Speculum
used for obtaining 2. Ayer‟s spatula = rotate 3600
specimen for 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 Honeycomb appearance

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cells Similar appearance to parabasal cells
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/ MI = 0/20/80
estrogen therapy
Quality Assurance
3 copies/report 1. Doctor
2. Patient = original copy
3. File
Reports Surgical pathology report
Cytopathology report
Autopsy report
Signatories Request forms = patient‟s doctor
Result forms = pathologist
Turnover of results Surgical pathology & cytology = 24 hrs
Frozen section = 5-15 mins
Autopsy report = 1 week (Autopsy procedure: 24 hrs)
Storage Specimen (tissue) = 1 month to 1 year
Tissue blocks (paraffin) = 3 to 10 years
Slides = indefinite
Suggested Guidelines for Record and Specimen Retention (Henry, 21st Ed.)
Records
Requisitions 2 years
QC 2 years
Instrument maintenance 2 years
BB QC 5 years
BB employee signatures 10 years
BB donor/recipient Indefinitely

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records
Reports
Clinical pathology lab 2 years
reports
Surgical pathology (and 10 years
BM) reports
Cytogenetics reports 20 years
Autopsy forensic Indefinitely
reports
Specimens
Serum/other body 48 hours
fluids
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

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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
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
available medico-legal cases & may authorize the pathologist to
proceed w/ an autopsy
The coroner has 1. All natural deaths occurring in the hospital w/in 24 hrs of
authority in the admission, unless the case was attended by a private
following cases 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
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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
pronouncement of death 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
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 1. irreversible cessation of circulation & respiratory
association & national functions
conference of 2. Irreversible cessation of all functions of the entire brain,
commission of uniform including the brainstem is dead
state laws legislative
definition of death
(1980)
American academy of Death:
neurology 1. Coma
2. Absence of the following:
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- 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
Technique of Rokitansky 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
Technique of Ghon En bloc technique
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Technique of Letulle En masse technique
♫ En masse:
- All organs of thoracic, abdominal, & pelvic are removed at
the same time
- Sweeping of all organs
Autopsy: Larynx  Very popular, easy to do, convenient
Rectum Part of consent: organs should be retained completely or
partially
Organs  set aside later
Body  undertaker of the body
Fresh Tissue Examination
Teasing/Dissociation Tissue specimen  Watchglass (isotonic solution)  BF/PC
microscope
Crushing/squash Tissue (<1mm)  Sandwich bet. 2 slides/coverslip  Vital
preparation stain
Smear preparation Spread lightly over a slide (wireloop/applicator)
Frozen Section
Frozen section (-) Fixative
Freezing of unfixed Best frozen section
tissue
Freezing of fixed tissue To localize hydrolytic enzymes & other antigens
Formal (formol) calcium Derivative of formaldehyde
Fix at 4‟C for 18 hrs
Commonly used methods Liquid nitrogen = most rapid
of freezing Isopentane cooled by liquid nitrogen
CO2 gas
Aerosol sprays
Staining methods “PATH”
(frozen sections) Polychrome methylene blue
Alcoholic pinacyanol
Thionine
H & E = progressive, no decolorizer
H&E a. Progressive
- w/o decolorizer
- for frozen sections
b. Regressive
- w/ decolorizer (acid-alcohol)
- for routine histology staining
Freeze-drying w/o use of any chemical fixative
♫ Quenching: rapid freezing (-160‟C)
♫ Sublimation: removal of H2O in the form of ice (-40‟C) –
vacuum
Freeze-substitution Similar to freeze drying but:
Frozen tissue  Rossman‟s formula/1% acetone
Dehydrated in absolute alcohol
Cold knife procedure Any microtome
Uses CO2
Knife: -40 to -60‟C
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Tissue: 5 to -10‟C
Environment: 0 to -10‟C
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 1st and most critical step
1‟ aim: preserve cell (life-like)
2‟ aim: harden & protect tissues
Most important: stabilization of proteins
pH 6.0-8.0
Temperature Room temp = Surgical specimen
0 to 4‟C = EM and Histochem.
Microanatomical General microscopic study of tissues
fixatives a. 10% Formol saline
b. 10% NBF
c. Heidenhain‟s SuSa
d. Formol sublimate (formol corrosive)
e. Zenker‟s solution
f. Zenker-formol (Helly‟s)
g. Bouin‟s solution
h. Brasil‟s solution
Cytological Specific parts of the cell
Fixatives a. Nuclear fixatives: w/ glacial acetic acid –
destroys mitochondria & golgi bodies (pH ≤4.6)
b. Cytoplasmic fixatives: w/o glacial acetic acid
c. Histochemical fixatives: preserves chemical
constituents
Nuclear fixatives “BFNCH”
Bouin‟s
Flemming‟s w/ acetic acid
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Newcomer‟s
Carnoy‟s
Heidenhain‟s SuSa
Cytoplasmic “HORFF”
fixatives Helly‟s
Orth‟s
Regaud‟s
Flemming‟s w/o acetic acid
Formalin w/ post chroming
Histochemical “FANA”
fixatives 10% Formol saline
Absolute alcohol
Newcomer‟s fluid
Acetone
Aldehyde Fixatives
Formaldehyde Concentrated solutions should not be neutralized
(explosion)
Stock solution: 37-40%
Working solution: 10% (no buffer: unstable)
Formalin pigments:
a. Paraformaldehyde
- White crystalline precipitates
- Due to prolonged standing
- Removed by: 10% METOH/filtration
b. Acid formaldehyde hematin
- Brown/black granular deposits that may obscure
microscopic details
10% Formol saline CNS
10% NBF Best general tissue fixative
Best fixative for tissue containing iron granules
w/ double phosphate buffer
1 mm/hr = rate of tissue penetration
Formol-Corrosive w/ HgCl2
(formol sublimate)
Glutaraldehyde EM
Karnovsky‟s EM: electron histochemistry & electron
paraformaldehyde immunocytochemistry
- glutaraldehyde
Acrolein Mixture w/ formaldehyde/formaldehyde
Formol-calcium Lipids (frozen section)
Fixatives
Mercuric Chloride Tissue photography
For Trichrome stain (excellent)
Produce black granular deposits except SuSa
“BOSCHZZ”
a. B5 = for BM biopsies
b. Ohlmacher‟s
c. Schaudinn‟s
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d. Carnoy-Lebrun
e. Heidenhain‟s SuSa = (-) black pigments
f. Zenker‟s = recommended for trichrome staining
g. Zenker-formol (Helly‟s) = pituitary gland, BM, &
blood containing organs
Heidenhain‟s SuSa Su = sublimat (HgCl2)
Sa = saure (acid)
HgCl2 Shrinks tissues
G.HAc Swells tissues, counteracts HgCl2
De-zenkerization Removal of mercuric deposits
H2O  I2  H2O  Sodium thiosulfate  H2O
Chromate “ROCK”
fixatives a. Regaud‟s (Moller‟s) = chromatin, mitochondria,
mitotic figures…
b. Orth‟s = for Rickettsia, tissue necrosis
c. Chromic acid = preserves CHO
d. K2CrO4 = mitochondria (if acidified, fixes
chromatin bodies & chromosomes but destroys
mitochondria)
Chromate Fine, yellow brown
pigments
Lead fixatives Used in 4% aqueous solution of basic lead acetate
For acid MPS and mucin
Picric acid Highly explosive when dry
fixatives Excessive yellow staining of tissues
Picrates  Protein  Ppt. (H2O soluble)  Add 70%
ETOH  Insoluble
Never wash in H2O before dehydration
For glycogen (excellent)
a. Bouin‟s = for embryos, Masson‟s trichrome stain,
glycogen
b. Brasil‟s alcoholic picroformol = less messy than
Bouin‟s, glycogen (excellent)
Glacial acetic acid Solidifies at 17‟C
Fixes & precipitates nucleoproteins, chromosomes,
& chromatin material
Most commonly combined w/ other fixatives
Alcoholic Disadvantage: polarization (glycogen granules 
fixatives poles/ends of the cells)
“MEICAN”
a. Methanol = BM & blood smears
b. Ethanol = preserves but does not fix glycogen
(Disadv: polarization)
c. Isopropanol = for touch preparations
d. Carnoy‟s = most rapid (1-3 hrs) | for chromosomes
| Dx: rabies (acetone)
e. Alcoholic formalin (Gendre‟s) = sputum
f. Newcomer‟s = for MPS | nuclear & histochemical
lec.mt 04 |Page | 271
fixative
Osmium tetroxide Inhibits hematoxylin
(Osmic acid) Produce black precipitate crystals (osmium oxide)
For lipids
a. Flemming‟s = permanently fixes fat, for nuclear
structures (excellent)
- Fixative & decalcifying agent (chromic acid)
b. Flemming‟s w/o acetic acid = for mitochondria
Trichloroacetic Precipitates proteins
acid Swelling effect  counteract shrinkage by other
fixatives
Weak decalcifying agent (softening effect)
Acetone Recommended for H2O-diffusible enzymes
(phosphatases, lipases)
Rabies
Heat fixation Bacteriologic smears
Microwave: 45-55‟C
Underheating: poor sectioning
Overheating (>65‟C): vacuolation, overstained
cytoplasm
2‟ fixation Placing an already fixed tissue in a 2nd fixative
Post- Primarily fixed tissue  2.5-3% K2CrO4 (mordant)
chromatization
Washing out Removing excess fixative
a. Tap H2O = remove excess chromates, formalin,
osmic acid (NOT Bouin‟s)
b. 50-70% alcohol = wash out excess picric acid
(Bouin‟s)
c. Alcoholic I2 = remove excess mercuric fixatives
EM fixatives Glutaraldehyde
PtCl3
PtCl3 – formalin (Zamboni‟s)
AuCl
Osmium tetroxide
10% NBF = acceptable but not recommended
Stains (EM) “PUL”
1. PTA = 1st general stain
2. Uranyl acetate = Best
3. Lead
Factors that Affect Fixation of Tissues
Retarded by:
Size & thickness
(+) Mucus Prevents complete penetration of fixative
Wash w/ NSS
(+) Fat Fatty tissues: cut in thin sections, fixed longer
(+) Blood Flush out w/ NSS  fix
Cold temperature Inactivates enzymes

lec.mt 04 |Page | 272


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 Fixed ASAP
specimens If not possible  refrigerate
If placed in NSS Autolysis may occur before fixation
during operation
If tissues are Avoid slow freezing (ice crystal formation)
refrigerated Repeated freezing & thawing  destroy organelles,
release enzymes…
Not more than Size of tissues
5mm thick 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 To prevent: place in a petri dish w/ moistened filter
small tissue 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- Do not use water
containing tissues Glycogen is water-soluble
Hard tissues Cervix, uterus, fibroids, hyperkeratotic skin,
fingernails
Wash in running water overnight  immerse in 4%

lec.mt 04 |Page | 273


aqueous phenol for 1-3 days (Lendrum‟s method)
Difficulties Encountered because of Improper Fixation
Problem Cause
Failure to arrest early cell Failure to fix immediately (tissue
autolysis was allowed to dry before fixing)
Insufficient fixative
Removal of substances soluble in Wrong choice of fixative
fixing agent
Presence of artifact pigments on Incomplete washing of fixative
tissue sections
Tissues are soft & feather-like in Incomplete fixation
consistency
Loss/inactivation of enzymes Wrong choice of fixative
needed for study
Shrinkage & swelling of cells & Overfixation
tissue structure
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 Brown/black granules “SAKaL”
formaldehyde a. Saturated picric acid
hematin b. Alcoholic KOH
c. Kardasewitsch method
d. Lillie‟s method
Mercuric chloride Black granules Alcoholic iodine
pigment
Chromate pigment Fine, yellow brown Acid-alcohol
Osmium tetroxide Black precipitate Cold H2O
pigment crystals
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 Acids
agents Chelating agents (EDTA/versene)
Ion exchange resins
Elec. ionization (electrophoresis)
HNO3 Most common
a. Perenyi‟s = tissue softener & decalcifying agent

lec.mt 04 |Page | 274


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 Hastens decalcification by removing calcium ions
resins from formic acid-containing decalcifying solutions
Electrophoresis Ca2+ are attracted to negative electrode (cathode)
Measuring extent Physical method
of 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- Removal/neutralization of acid from the tissues
Decalcification after decalcification
Lithium carbonate or sodium bicarbonate solution
Tissue softeners 4% phenol
Molliflex = tissues appear swollen & soapy
2% HCl
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 Dehydration w/ dioxane
method
Weiseberger‟s
method
Cellosolve Ethylene glycol monoethyl ether
Combustible and toxic
Triethyl --

lec.mt 04 |Page | 275


phosphate
Additives to a. 4% phenol + 95% ETOH = softener
dehydrating b. Anhydrous CuSO4 (Last ETOH bath)
agents - both dehydrating agent & indicator of H2O
content of 100% ETOH
- (+) H2O = White  Blue
Methods of 1. Anhydrous CuSO4 method
determining 2. Xylene  Milky
incomplete
dehydration
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
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

lec.mt 04 |Page | 276


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 Wax impregnation under negative pressure (hasten
embedding 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 Indicates that the paraffin wax should be changed
clearing agent
Embedding
Orientation Arranging in precise positions in the mold,
microtome & slide

lec.mt 04 |Page | 277


Oven 5-10‟C above the MP of wax (Impregnation: 2-5‟C
above)
Blocking-out Molds
Leuckhart‟s 2 L-shaped strips
embedding mold Adjustable
Compound Several compartments
embedding unit
Plastic embedding Special stainless steel base mold fitted w/ a plastic
rings & basse embedding ring (block holder)
mold
Tissue Tek Warm plate
Cold plate (-5‟C)
Disposable 1. Peel-away: perfect even block w/o trimming
embedding molds 2. Plastic ice trays: ordinary refrigerators
3. Paper boats: cheap & easy to make
Celloidin/Nitrocell Bones, teeth, eyes
ulose method Bell jars: control evaporation
Double embedding 1st: celloidin
method 2nd: paraffin
Brain
Recall Temperatures
Cold knife Knife = -40 to -60‟C
procedure Tissue = 5 to -10‟C
Environment = 0 to -10‟C
Cryostat (Cold -18 to -20‟C
microtome)
Fixation Surgical specimen: room temp
HC & EM: 0-4‟C
Freeze-drying Quenching: -160‟C
Sublimation: -40‟C
Algor mortis 7‟F/hr (3.89‟C/hr)
Formol calcium 4‟C
fixation
Impregnation 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
Embedding 5-10‟C above MP of wax
Flotation water 6-10‟C below MP of wax (45-50‟C)
bath
Trimming
Trimming Removing excess wax after embedding
Ideal: four-sided prism/truncated pyramid
Microtomy
Routine histopath. 4-6μm
(Rotary)
Freezing 10-15μm
EM (Ultrathin) 0.5μm

lec.mt 04 |Page | 278


Rocking/Cambridg Trefall
e microtome Simplest
Rotary/Minot Minot
microtome Most commonly used for paraffin embedded tissues
Sliding microtome Adams
Most dangerous (movable knife)
a. Base-sledge
- For all forms of media
- Block: moving
- Knife: stationary
b. Standard sliding
- Block: stationary
- Knife: moving
Vibrotome For unfixed, unfrozen tissues
For enzyme demonstration
Ultrathin EM
microtome Diamond knives or broken plate glass
Freezing Queckett
microtome
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
lec.mt 04 |Page | 279
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 Alum hematoxylin
forces
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
Hematoxylin ---(Ripening)---> Hematein (active
coloring substance)
Lake Tissue-Mordant-Dye complex
Oxidizing agents H2O2
HgO2 = Harris‟
K2MnO4
Na perborate
Na iodate = Mayer‟s, Ehrlich‟s, Gill‟s
Alum hematoxylin 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)
Iron hematoxylin 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
Tungsten a. Mallory‟s PTAH
hematoxylin - Mordant = sunlight/K+
- Stain fibrin
lec.mt 04 |Page | 280
Copper Spermatogenesis
hematoxylin
Eosin (Eosin Y) 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
Coplin jar Holds 5-9 slides
Slotted staining Holds 5-19 slides
dishes
Metal/glass Holds 10-30 slides
staining racks/
carriers
H & E staining 1. Xylol (2) = deparaffinization
steps 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
Pap Smear Hematoxylin = nuclear stain
staining 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
lec.mt 04 |Page | 281
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 Slide  Xylene (24hrs) or gently heat until
sections 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 mounting media
2. If replacement not possible, the section (if
intact) may be transferred to another slide:
Broken slide  Xylene (rem. coverslip)  incubate
(rem. mountant)  6 parts butyl acetate + 1 part
lec.mt 04 |Page | 282
durofix  incubate (mixture  film)  Cut the
film around the section  Cold H2O until the film &
section float off  Film w/ section  mount on a
clean slide  incubate  butyl acetate  xylene 
mount

Shortcut: “Xi6B1DiCuCoFSMiBXM”
Broken slide  Xylene  Incubate  6 Butyl
acetate + 1 Durofix  Incubate  Cut film  Cold
H2O to float film & section  Film w/ section 
mount  incubate  butyl acetate  xylene 
mount
Ringing Sealing the margins of the coverslip
Prevent escape/evaporation of fluid
Immobilize the coverslip
Prevent sticking of slides
a. Kronig cement = 2 parts paraffin + 4-9 parts
colophonium resin
b. Durofix (cellulose adhesives)
Immunohistochemistry
Enzyme Trypsin & protease = most commonly used
histochemistry
IgG Most commonly used antibody
Polyclonal Rabbits (1‟) > Goat (2‟) > Pig (3‟) > Sheep (4‟) > Horse
(5‟) > Guinea pig (6‟)
Monoclonal Mice
Epithelial Tumor Markers
(+) CK 7 “LUBO” = paired
(-) CK 20 Lung
Uterus
Breast
Ovary
(+) CK 20 Stomach
(-) CK 7 Colon
(+) CK 7 Transitional cell carcinoma of the bladder
(+) CK 20 Mucinous ovarian tumor
(-) CK 7 HCC
(-) CK 20 RCC
SCC
Thyroid carcinoma
Prostatic adenocarcinoma
EMA (Epithelial (+) carcinoma “BuLK” = paired
membrane Breast
antigen) Lungs
Kidney
CEA Oncofetal antigen
GI carcinoma
Differentiates adenocarcinoma (+) & mesothelioma
lec.mt 04 |Page | 283
(-)
TTF-1 (Thyroid Differentiates lung adenocarcinoma & mesothelioma
Transcription (+): Thyroid, lung, neuroendocrine tumors
Factor)
PSA Prostate cancer
Intermediate Filament Markers
Actin Smooth muscle
Skeletal muscle
Cardiac muscle
Vimentin Melanomas
Schwannomas
Desmin Leiomyoma (smooth muscle)
Rhabdomyosarcoma (skeletal muscle)
GFAP (Glial Astrocytoma
Fibrillary Acidic
Protein)
NF Neuroblastoma
(Neurofilament) Ganglioneuromas
Neuroma
Chemodectoma
Pheochromocytoma
S100 protein Low MW Ca2+-binding protein
CNS glial cells, Schwann cells
Neuroendocrine Markers
NSE (Neuron- Strong evidence of neural/neuroendocrine
specific enolase) differentiation
Others Chromogranin
Synaptophysin
Germ Cell tumor markers
HCG Synthesized by syncytiotrophoblasts
Choriocarcinoma
AFP Endodermal sinus tumors showing yolk sac
differentiation
PLAP (Placenta- Germinomas
like ALP)
Mesenchymal Tumor Markers
Myogenic tumors Myo-D1
Myoglobin
Myogenin
Fibrohistiocytic --
tumors
Vascular tumors Factor VII-related antigen
CD31
UEA: Ulex europaeus I
Melanomas --
Lymphomas LCA: Leukocyte common antigen (CD45)
Cell Proliferation Markers

lec.mt 04 |Page | 284


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 A.k.a. “built-in control”
control Contains the target antigen
Other Topics
Faults During Tissue Processing
Brittle/hard
tissue

Clearing agent  Incomplete dehydration


Milky
On trimming, Insufficient impregnation
tissue smells of
clearing agent
Tissue is opaque Insufficient clearing
Tissue shrinks Insufficient dehydration
away from wax Incomplete clearing & impregnation
Tissue is soft Incomplete fixation
when block is
trimmed
Air holes on Incomplete impregnation
tissue
Wax appears Contaminated wax
crystalline Block not cooled rapidly enough
Paraffin block is Insufficient paraffin impregnation
moist & crumbles
Sections fail to Surface & edges of block not parallel
form ribbons Wax too hard
Knife tilted too much
Thick sections
Dull knife
Sections roll up on Blunt knife
cutting… adhere &
get broken Dirty knife edge
against the knife
edge
Ribbon is curved, Irregular knife edge
crooked, or Edges of block are not parallel
uneven Knife not parallel to the block

lec.mt 04 |Page | 285


Impure paraffin
Sections are Blunt/dull knife
compressed, Block is warm & soft
wrinkled or Knife edge coated w/ paraffin
jammed Thin sections
Microtome screw is loose
Tilt: vertical
Sections are Bevel of knife is lost
squashed Incorrect sharpening
Hole in section Bubble/dirt
Hard spot in the tissue (Ca2+)
Sections of
unequal thickness Screw/holder is loose
Large & hard blocks
Sections adhere Static electricity
to knife or other Dirty knife edge
parts of the Dull knife edge
microtome
Ribbon is split Nicks/damage on knife
Dirty embedding
Dirty knife

Chatters are seen Knife vibrates (hard tissue)

Section: Blunt knife


sometimes thin &
thick Knife/block holder is loose
Frozen tissue Inadequate freezing
crumbles & comes
off when the
block holder when
cut
Frozen tissue Tissue is frozen too hard
chips into
fragments

lec.mt 04 |Page | 286


Stains
Stain Substance (+) Color/Result Comments
Stained
PAS CHO, Glycogen, PAS Basic fuchsin:
Mucins, (+):Magenta red essential
Bacteria & component of
Fungi, basement Schiff reagent
membrane
PAS w/ diastase Glycogen Red Method of
ctrl choice for
glycogen
staining
Best Carmine Glycogen Bright red Selective &
highly specific
for glycogen
Langhan‟s iodine Glycogen Mahogany brown Obsolete
method 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: hematoxylin
magenta
Gomori‟s aldehyde Acid MPS Sulfated
fuchsin stain Sulfated mucins mucins: purple
Carboxylated Carboxylated
mucins mucins: blue
Mucicarmine stain Cryptococcus Mucin: red Avoid Ehrlich‟s
neoformans hematoxylin
Mucins
Colloidal Acid mucins Dark blue
(Dialyzed) iron
technique
Acridine orange Acid Acid MPS: black Lasts for only 2
mucins/MPS Fungi: greenish hrs
Fungi red
fluorescence
Sudan black Lipids Blue black
Sudan IV Lipids (TAG) red Most commonly
(Scharlach R) used stain
Oil red O Lipids Brilliant red
Osmium tetroxide Lipids Black
Nile blue sulfate Neutral fat = Pinkish red Nile blue:
method Cholesterin = Light red preliminary
esters = Light red indicator of the
Cholesterin = Deep blue to type of lipid
fatty acids violet present
lec.mt 04 |Page | 287
Fatty acids & = Light blue -Red oxazone
soap (dissol. neutral
Cerebrosides lipids)
-Blue oxazone
(reacts w/ PL
and FFA)
Toluidine blue- Sulfatide Metachromatic
acetone mtd red-brown or
yellow
Borohydride- Gangliosides Red
Periodic-Schiff
method
Alkaline fast- Histones Green Fast green
green method Protamines stains basic
groups in
tissues
Peracetic acid- Cystine Blue-green
Alcian blue Cysteine
Sakaguchi‟s test Arginine Orange-red Uses Milton
reagent
Gomori calcium Alkaline Brownish-black
method phosphatase
Gomori lead Acid Black Substrate: β-
method phosphatase glyceroPO4
Lead method 5‟-nucleotidase Blackish brown
deposits
Metal ATPase Dark brownish- For skel. muscle
precipitation black ppt biopsies
Calcium cobalt ATPase Cobalt For skel. muscle
method phosphate ppt biopsies
α-naphthyl Nonspecific Reddish brown
acetate method esterase
Stain Substance (+) Color/Result Comments
Stained
Indoxyl acetate Nonspecific Blue
method esterase
Tetrazolium Monoamine Bluish black
method oxidase
Feulgen technique DNA Red-purple Most reliable &
specific
histochemical
staining
technique for
DNA
Contains
Schiff‟s reagent
Methyl green- RNA RNA (nucleoli):
pyronin DNA red
lec.mt 04 |Page | 288
DNA
(chromatin):
green
Gomori‟s silver Reticulin fibers black Reticulin =
impregnation stain Argyrophilic
(silver stain)
Van Gieson‟s stain Collagen = Pink/deep red Contains acid
Muscle, = Yellow fuchsin & picric
cytoplasm, RBC, acid
fibrin
Masson‟s Collagen & = Blue
trichrome stain mucus = Red
Muscle, RBC &
keratin
Mallory‟s aniline Collagen fibers, = Red (-) Fuchsin:
blue cytoplasm, Excellent &
fibroglia fibrils, colorful method
axon cylinders, of
neuroglia = Pale demonstrating
Elastic fibers pink/yellow CT fibers
RBCs, myelin = Yellow
sheets
Azocarmine CT Heidenhain‟s
Glomerular modification of
basement Mallory‟s aniline
membrane = Deep blue blue stain
Amyloid &
mucous colloid
Weigert‟s Elastic fibers Dark-blue/blue-
black
Verhoeff‟s Elastic fibers Black
Taenzer-Unna- Elastic fibers Dark-brown
Orcein mtd
Krajian‟s Elastic fibers = Bright red Rapid method
technique Fibrin & CT = Dark blue
RBC = Orange-yellow
Martius-Scarlet- RBCs = Yellow Early fibrin =
Blue Muscle = Red yellow
Collagen = Blue Old fibrin = blue
Fibrin = Red
Mallory‟s PTAH Fibrin, muscle = Dark blue
striations,
neuroglia, = Blue
amoeba = Lighter blue
RBCs = Deep
Myelin brownish-red
Collagen,
osteoid,
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cartilage,
elastic fibers
Congo red Amyloid Red
Methyl violet- Amyloid Purplish red
crystal violet
method
Thioflavin-T Amyloid Yellow
fluorescent fluorescence
staining
Modified Gomori‟s Muscle fibers = Red
Trichrome stain Collagen = Green
Lissamine fast Muscles, RBC = Red
red Collagen = Yellow
Stain Substance (+) Color/Result Comments
Stained
Schmorl‟s Picro- Lacunae & = Dark brown-
Thionin method canaliculi black
Bone matrix =
Yellow/brownish
-yellow
Bielschowsky‟s Neurofibril, = Black on a
technique axons & grayish BG
dendrites
Neuroglia & = Lightly
collagen stained
Bodian‟s stain Nerve fibers & Diagnosis of
nerve endings Alzheimer‟s
disease
Sevier-Munger Peripheral = Black
technique neuritis = Black
Axons = Light brown
Myelin sheath = Black
Neuritic plaques
& tangles = Black
Argentaffin
granules
Toluidine blue Nissl granules & Deep blue
nucleoli
Polychrome Nissl granules & Deep blue
methylene blue nucleoli
Thionine Nissl granuls & Purple
nucleoli
Cresyl fast violet Nissl substance = Purple-dark Nissl granules:
Neurons blue a.k.a. Tigroid
= Pale purple substances
blue
Weigert-Pal Myelin sheath Blue black
technique
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Luxol fast blue Myelin Blue-green
Weil‟s method Myelin Black
Cajal‟s gold Astrocytes Black on a light
sublimate method brownish BG
Perl‟s Prussian Hemosiderin Deep blue
blue
Gomori‟s Prussian Iron pigments Bright blue
blue
Turnbull‟s blue Ferrous iron Blue
(Hemosiderin)
Benzidine- Hemoglobin & Dark blue
nitroprusside oxidase granules
stain
Mod. Fouchet‟s Bile pigments Emerald to blue
technique green
Gmelin technique Bile & Blue-purple then
hematoidin 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 melanin,
method argentaffin
cells,
chromaffin,
thyroid colloid
Gomori‟s aldehyde Lipofuscin Purple
fuchsin
Mallory‟s fuchsin Hemofuscin Red
stain
Masson Fontana Melanin = Black Argentaffin
technique Argentaffin cell = Black reaction:
granules melanin reduces
ammoniacal
silver solutions
w/o use of a
reducer
Von Kossa‟s silver Calcium Black
nitrate method
Lindquist modified Copper Red to orange-
rhodanine red
technique

Stain Substance (+) Color/Result Comments


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Stained
Gram-Twort stain Gram (+) = Blue-black
organisms = Pink-red
Gram (-) = Green
organisms = Black
RBCs
Elastic fibers
Brown & Brenn Gram (+) = Blue
method bacteria = Red
Gram (-)
bacteria
Wade-Fite M. leprae Golden yellow
technique
Toluidine blue H. pylori Dark blue
against blue BG
Cresyl violet H. pylori Blue-violet
acetate mtd
Dieterle method L. pneumophila & Dark brown to
spirochetes black
Levaditi‟s method Spirochetes Black on a
yellowish BG
Modified Steiner Spirochetes, Black
& Steiner Donovan bodies,
technique fungi, bacteria
Warthin-Starry Spirochetes Black
method
Grocott Fungi = Sharply
Methenamine outlined I black
Silver Mucin & = Gray-black
glycogen = Old rose
Mycelia & = Yellow
hyphae
RBCs
Lendrum‟s Viral inclusions Bright red
phloxine-
tartrazine method
Orcein method HBsAg Brown-black
Giemsa stain Bacteria = Blue Recommended
Mast cell = Deep blue for blood and
granules = Red BM parasites,
Eosinophilic = Blue inclusion
granules = Pink conjunctivitis,
Nuclei Toxoplasma,
Cytoplasm spirochetes &
other bacteria
In situ Most sensitive technique for identifying DNA
hybridization
PCR DNA amplification
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Chondrocalcinosis Pseudogout
Kardasewitsch Pigment removal
method 70% ETOH + 28% NH3 water
0.1% urea + 5% Remove yellow color of HNO3
NaSO4
Metastasis Most definitive of malignancy
Degree of Most reliable indicator of prognosis of malignant
localization tumors
Dunn-Thompson Hgb = emerald green
K2MnO4 Removes excess melanin
H2O2
Helly‟s Contains formalin, K2CrO4 and HAc
Formalin Fixative for CNS (gold/silver stain)
ammonium
bromide
Alcohol as 1‟ Increased tissue shrinkage
fixative
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
pH >6.0 (formalin) Prevent brown microcrystalline deposits in H & E
stain
Universal solvents Both dehydrates & clears
Soft paraffin For thick sections
Weigert‟s Not easily decolorized w/ acidic solutions
hematoxylin
Natural resins Inherently acidic
(mounting)
Formalin-alcohol Microincineration
Churukian-Schenk Substance that can bind silver but need a chemical
technique reducer
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 Calibrate pH meter to 7.0
stain
Iris diaphragm Increase amount of light
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Substage Adjust to focus the image of the substage
condenser 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 a. Direct staining = w/o mordant
presence of a b. Indirect staining = w/ mordant: serves as a
mordant link/bridge between the tissue & the dye
According to the a. Progressive = w/o differentiator/decolorizer
presence of b. Regressive = w/ differentiator/decolorizer
differentiator *1‟ stain = acidic (decolorizer: basic)
*2‟ stain = basic (decolorizer: acidic)
According to the a. Orthochromatic = “ortho”: correct/same | same
resultant color color = dye & tissue
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
b. Axons = conduct impulses away from the cell body
Criteria for the Marked progesterone effect
diagnosis of At least 50% of intermediate cells in clusters
normal pregnancy At least some typical pregnancy cells present
<30% of matured superficial cells
Doderlein-filled dirty BG
Staining solutions a. Hematoxylin = dark purple to black
used in Pap‟s b. OG-6 = orange w/ a hint of green
staining method c. EA 36/50 = olive green w/ a hint of brown & red
(Macroscopic)
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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 Class I
lesion/malignancy
Atypical ASCUS Class II
HPV L-SIL Class II
Mild dysplasia L-SIL Class II
Moderate H-SIL Class III
dysplasia
Severe H-SIL Class III
dysplasia
Carcinoma in H-SIL Class IV
situ
Invasive Invasive carcinoma Class V
carcinoma
Usual
Microtom Microto
Lengt Description Tissues embedding in
e Knives me
h
Plane 25 One side Less concave: celloidin- Sliding
concave mm flat, other is impregnated tissues
concave More concave: paraffin
embedded tissues Base-
sledge
Rotary
Rocking
Biconcave 120 Both sides Paraffin Rotary
mm are concave
Plane 100 Both sides Frozen sections Sliding

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wedge mm are straight Hard, tough tissue specimen Base-
(paraffin) sledge
Carmine Chromatin stain
Best Carmine Carmine + Aluminum chloride = For glycogen
Mucicarmine Carmine + Aluminum hydroxide = For C. neoformans
and mucin
Picrocarmine Carmine + Picric acid = for neuropathological studies
Duke‟s staging for One of the most frequently applied for staging
neoplasia of the individual tumors
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 Desquamated cells
cytology 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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