MTAP2: HISTOPATHOLOGY

Mr. Rene Jesus Dinglasan | Week 10

HISTOPATHOLOGIC TECHNIQUES TOTAL 100%

Histopath, MT Laws, Related Laws, and Code of Ethics (Table
of Specifications for the Licensure Examination)
● Total weight: 10% minor subject BASIC HISTOPATHOLOGY
TOPICS WEIGHT (%) I. INTRODUCTION
● Histopathology: study of abnormal tissues
1 Histology and Pathology 10% ● Pathology: study of disease
● Derived from two greek words:
Terminologies 4% ○ “Pathos”: suffering
○ “Logos”: study
Etiology of disease 2% *Cytology - study of cells
IMPORTANT PERSONALITIES
Signs, symptoms and course of disease 2%
Paldwell Trefall
Cellular and tissue changes 2%
John Thomas Quekett
2 Histopathologic techniques and procedures 35% Inventors of microtome
George Adams
Preservation and handling of specimen 10%
Charles Minot
Tissue processing and procedures 15%
Hippocrates Father of Medicine
**Routine-manual (7%)
Giovanni Battista Father of Modern Anatomical
**Special-frozen section, microwave (3%)
Morgagni Pathology

Staining 10%
Rudolf Virchow Father of Cellular Pathology

**Routine (5%)
George Papanicolaou Father of Exfoliative Pathology

**Special (immunohistochemistry) (5%)

Karl Landsteiner Father of Blood Transfusion

3 Cytological techniques and procedures 8%

Julius Cohnheim Introduced frozen section examination

Preservation and handling of specimen

II. INFLAMMATION
Processing ● From the Latin word “inflammare”: to set a fire
● Protective response of the tissues of the body to
**Manual (2%) irritation or injury
● It is composed of a series of physiologic and
**Automation (2%) morphologic changes in the blood vessels, blood
components and surrounding connective tissues for
Staining the purpose of protecting the body against injury

4 Autopsy 2%
5 Cardinal or Primary Signs of Inflammation
Terminologies 1%
Rubor (redness) Due to arteriolar and capillary dilatation
with increased rate of blood flow towards the
Handling, processing and documentation 1%
site of injury

5 Quality assurance 10%

Tumor (swelling) Due to increased capillary permeability →
causing extravasation of blood fluid
6 MT laws 10%
Calor (heat) Due to transfer of internal heat to the
7 Laboratory management 10% surface or site of injury, brought about by
increased blood content
8 Related laws 10%
Dolor (pain) Due to pressure upon the sensory nerve by
9 Code of ethics including Bioethics 10% exudate or tumor

AT | DP | ST | MS | CP | PP | RC | CM 1
 Functio laesa Destruction of the functioning units of the Examples:
(diminished tissue ● Vascular atrophy
function) ● Pressure atrophy
● Starvation or Hunger atrophy
● Atrophy of disuse
Classifications of Inflammation (according to duration): ● Exhaustion atrophy
● Endocrine atrophy
Acute ● a.k.a. Exudative Inflammation
inflammation ● Usually, but not necessarily, of sudden 2. Progressive changes: organ or tissue larger than normal
onset
● Vascular and exudative Hypertrophy ● Tissue size increases because of size
● Predominantly polymorphonuclear increase of individual cells
neutrophils cells (PMNs) Example:
● When this fails to subside within several ● Increase in the size of the uterus during
weeks → chronic inflammation pregnancy (to accommodate the fetus)

Subchronic ● Represents an intergrade between acute
inflammation and chronic inflammation (stage where
the acute inflammation transforms to
chronic inflammation)
Chronic ● Persistence of the injuring agent for
inflammation weeks or years
● Vascular and fibroblastic
● Predominantly mononuclears
(macrophages, lymphocytes, plasma
cells) but PMNs may also be present
III. CHANGES IN CELLULAR GROWTH PATTERNS
1. Retrogressive changes: organ or tissues smaller
than normal
a. Developmental defects
Aplasia ● Incomplete or defective development of
tissue or organ
● Most commonly seen in one of paired
structures (kidneys, gonads, adrenals)
● Example: one of the two kidneys would
look like normal but they other would look
like a lump of fat
Agenesia ● Non-appearance of an organ
Hypoplasia ● Failure of an organ to reach its full,
mature size
● The organ did not grow or reach its mature
size in the first place
Atresia ● Failure of an organ to form an opening
Examples:
● Biliary atresia: bile duct did not form or the
bile duct is present but has no opening
● Bilirubin cannot be excreted → Stool is
white in color
b. Atrophy
● Decrease in the size of a normally mature tissue or organ
● Resulting from the decrease in the cell size or decrease in the total
number of cells comprising the tissue or organ
Physiologic ●
Occurs as a natural consequence of
maturation
Examples:
● Atrophy of the thymus during puberty
● At about 50 years old: atrophy of the brain
and sexual organs
Pathologic ● As consequence of disease
Hyperplasia ● Tissue size increases because of increase in
the number of cells making up the tissue
Two categories (Hyperplasia):
A. Physiologic Hyperplasia:
● Increase in the size of the uterus during
pregnancy
● Glandular proliferation → female breast
during lactation
B. Pathologic Hyperplasia:
● Typhoid fever: hyperplasia of the lymphoid
follicles and peyer’s patches of the intestines
3. Degenerative Changes
a. Metaplasia
● Transformation of one type of adult cell to
another type of adult cell
○ One type → Another type
Of adult type ← adult cell
○ Reversible: can go back to original
form once the stimulus stops and
as long as there are no cancer cells
● Example:
○ Metaplasia in the trachea of chronic
smokers
- Normal: pseudostratified columnar
epithelial tissues that are ciliated
- Abnormal: stratified squamous
epithelium (lung area is not
protected, has no cilia,
microorganism or foreign
pathogens from environment can
enter into the lungs
b. Dysplasia
● Changes of one particular cell
○ One type → Changes in
Of adult type ← structural components
● Reversible: can go back to original form
once the stimulus stops and as long as there
are no cancer cells
c. Anaplasia
● Irreversible
● Transformation of one type of adult cell to a
more primitive cell type
○ Adult cells → More primitive cells
● Usually used as a criterion towards
malignancy
● May progress to cancer later on
d. Neoplasia
● Process of tumor formation
● Continuous abnormal proliferation of cells
without control (no purpose or function)
AT | DP | ST | MS | CP | PP | RC | CM 2
 particularly malignant neoplasm
Fibrous tissue Fibroma Fibrosarcoma
● Neoplasm: from greek word “new growth”
also referring to tumors (generally, usually)
Adipose/Fatty Lipoma Liposarcoma
○ Not all neoplasm are tumors
Cartilage Chondroma Chondrosarcoma
NEOPLASM
Bone Osteoma Osteogenic sarcoma
Part of Tumors
Blood Vessels Hemangioma Hemangiosarcoma
Parenchyma ● Active elements within the tumor (this
pertains to the tumor cells themselves) Hematopoietic cells Leukemia
● Heto yung mga tumor cells na mismo nag
proliferate doon sa loob ng tube Lymphoid tissue Lymphoma

Stroma ● Connective tissue framework where this Smooth muscle Leiomyoma Leiomyosarcoma
active elements are embedded
● Connective tissue framework where
Striated muscle Rhabdomyomas Rhabdomyosarcoma
parenchymal cells are found

Types of Tumor
According to the Capacity to produce Death B. Epithelial Tissue Tumors

Benign Tumor ● Usually, not deadly BENIGN MALIGNANT

● If an organ is already compromised
because of its growth, patients ay die Stratified squamous Squamous cell Squamous cell
● Suffix: “-oma” papilloma carcinoma
○ Example: Lipoma
○ Exemption: Some end with Glands and Ducts Adenoma Adenocarcinoma
“-oma” but are malignant tumors
(e.g., seminoma, melanoma, Renal Epithelium Renal tubular Renal cell carcinoma
hepatoma) adenoma
﹘ Other examples:
Hematoma and
Lymphoma Liver cells Liver cell adenoma Hepatocarcinoma
(Hepatoma)
Malignant ● Collective term for the malignant tumors or
Tumor neoplasm is Cancer (comes from the latin Neuroectoderm Nevus/Mole Melanoma
word “cancrum” meaning crab) (Melanocarcinoma)
○ Cancer cells are like crabs, they
are stubborn. Once they attach Testicular epithelium Seminoma
to the host, they are difficult to
remove.
○ There are other cases that Grading of Tumors
cancer cells are returning even ● “Grading”: grading of a cancer attempts to establish some
though the patient undergo estimate of its aggressiveness or level of malignancy
chemotherapy, proper treatment based on the cytologic differentiation of tumor cells and
and so on the number of mitoses within the tumor
● Usually deadly ● Note: Well-differentiated tumors as a rule are less
● If patient is treated early (stage I cancer)
malignant than undifferentiated tumors
may not be deadly
● Suffixes:
○ Differentiated cells: resemble to normal cells
○ “-sarcoma”: origin of ○ Undifferentiated cells: resembles to younger forms of
mesenchymal or connective cells
tissue
○ “-carcinoma”: origin of
epithelial tissue BRODERS CLASSIFICATION
● Ex. Lymphoma, Liposarcoma (collective
term for the malignant Connective Tissue; Differentiated cells Undifferentiated
malignant tumor of fat tissues or adipose) cells

According to Histologic Characteristics Grade I 100-75% 0-25%

Medullary Cells > supporting tissue Grade II 75-50% 25-50%

Scirrhous Connective tissue > cells Grade III 50-25% 50-75%

Grade IV 25-0% 75-100%

A. Mesenchymal or Connective Tissue Tumors
BENIGN
● Broders classification: acts as a guide to treatment
● Lower grade: amenable to Surgery
MALIGNANT ○ Can still be treated using surgery
● Higher grade: amenable to Radiation
AT | DP | ST | MS | CP | PP | RC | CM 3
 ○ Rarely done in certain cases; weakens ● Happens when cell detects that it is abnormal
patient (side effects) ● Mechanism of the body to prevent the proliferation
○ Other treatment options: use of Abs (highly of abnormal cells
specific) ● Ex. Cells in the bone marrow that may sometimes
be abnormal, if that marrow cell detects that it’s
Staging of Tumors/Cancers abnormal, it will kill itself so that the abnormal cell
● “Staging”: is based on the: will not proliferate
○ Size of the primary lesion b. Necrobiosis
○ Its extent of spread to regional lymph nodes ● Physiologic or normal death of cells
○ Presence or absence of metastases ● Cell dies because of old age
- Metastasis: singular form ● Ex. dead skin cells (outer layer of skin)
- Metastases: plural form c. Necrosis
● Metastasis ● Pathologic death of cells
○ Spread of cancer cells to distant areas of the
body
TYPE OF NECROSIS
- Cancer cells are dislodged and go
to the distant parts of the body
Coagulation ● Most common of the five
through the bloodstream Necrosis ● Demonstrates “tombstone formation”
- Cancer cells are like parasites. ● Upon the death of the cell, the
They obtain nutrients from the organelles get lost in the cell and only
patient’s body and grow bigger until outline of the cell remains → looks like
the patient dies slowly lapida/tombstone
● Content of the cell disappears, but
● Teratomas outline can still be seen
● Often seen in: myocardium, lungs,
○ Type of neoplasm
kidneys, and spleen
○ Compound tumors
○ Greek: “monstrous tumors”
Liquefaction ● Also known as Colliquative necrosis
- Looks like monsters Necrosis ● Characterized by pus formation
○ Tumor with normal tissue or organ ● Often seen in: brain
components that are inappropriate to
surrounding tissues (that’s why “monstrous”) Caseous ● Also known as Caseation necrosis
○ May contain hair, teeth, bones, and very Necrosis ● Characterized by the presence of yellow,
rarely eyeballs, torso, and hands “cheesy”, crumbly materials
● Often seen in:
Two major agencies concerned with the staging of ○ M. tuberculosis infection,
malignant disease are: ○ syphilis,
○ tularemia,
1. UICC: International Union Against Cancer ○ lymphogranuloma inguinale
● Devised TNM system of cancer staging
2. AJCS: American Joint Committee on Cancer Staging Gangrenous ● Histologic characteristic:
● Uses roman numerals (I, II, III, IV) in staging of Necrosis ○ Combination of the
cancers liquefaction and
● Combined grading and staging criteria (Stage I, II, III, coagulation necrosis
IV) ● Types:
● TNM system can be converted into numerical staging ○ Wet gangrene: usually
or vice versa caused by venous occlusion
(blockage)
○ Dry gangrene: usually
TNM System of Cancer Staging caused by arterial occlusion

● Developed by UICC Fat Necrosis ● Characterized by “chalky” white

● Applicable to all forms of neoplasia precipitates
● Seen in pancreatic degeneration:
T - Tumor (primary T1, T2, T3, T4: with increasing size of the ○ Pancreas: is the organ that
tumor) primary lesion produces amylase, and lipase
(fat metabolism)
○ Lipase: (needed for lipase
N - Nodes N0, N1, N2, N3: indicates progressively
metabolism) from
(Regional lymph advancing nodal disease
degenerated pancreas leaks
node involvement)
out → this enzyme destroys
the surrounding fats in the
M - Metastases M0 or M1: whether there are spread of cancer pancreas → fat necrosis
cells to the distant metastases part of the
body
B. Somatic death: death of the entire body
a. Primary changes: changes that occur during somatic
IV. DEATH death (C.R.C)
A. Cellular death ● Circulatory
a. Apoptosis ● Respiratory
● Programmed cell death ● Central nervous system failure
AT | DP | ST | MS | CP | PP | RC | CM 4
 b. Secondary changes: changes that occur after
somatic death (A.R.L.P.D.A: Aral Pa D Pa)
● Algor mortis:
○ First demonstrable change observed
○ Cooling of the body or cadaver after death
﹘ Panlalamig ng katawan ng tao
pagkatapos niya mamatay
○ Rate: 7℉ per hour ● Post mortem clot:
○ This is used to estimate how long the person ○ Differences between:
has been dead especially in the investigation
of death POSTMORTEM CLOT ANTEMORTEM CLOT
● Rigor mortis:
○ Stiffening of skeletal muscles after death “Chicken fat” in appearance Granular and friable
﹘ Pagtigas ng katawan or cadaver
○ First occur in the muscles of the head and in “Currant jelly” in appearance Not easily detached from the
blood vessel wall
the neck
● Livor mortis:
Assumes the shape of the blood Seldom assumes the shape of
○ Also known as Postmortem vessel where it came from blood vessel
lividity/postmortem suggillations
○ Purplish discoloration/lividity of skiN after Rubbery in consistency
death
○ Looks like ecchymoses Formed “After Death” (anyone Formed “Before Death” (may be
○ Recognize that it is livor mortis and not who dies will have this) the cause of death of the patient)
ecchymosis
○ Usually Livor mortis is seen at the back of ● Postmortem: after death ● Ante: before
the cadaver since usually people died at lying ● This are clot that may ● This are clot that forms
down position then the gravity will pull the appear in the body after before the death
RBC downward toward the back area of the death ● One probable reason
body ● This means they are why the patient died
○ Sometimes this livor mortis may occur natural or normal happen
for death
together with the Ecchymosis

Differentiate the Liver Mortis from Ecchymoses

- This matter especially if investigating the cause of death
LIVER MORTIS
(Postmortem lividity or
postmortem suggillations)
Happens because the heart
stopped pumping blood
Stagnant blood flow → gravity
pulls down the RBCs
Depends on where RBCs will
settle:
● If you die lying down,
discoloration will be at
the back
● If you die face down,
discoloration will be at
the front
General characteristics:
● Disappears on pressure
(reappears purplish
discoloration when
pressure is released)
General characteristics:
● On incision, oozing of
blood is observed
● Desiccation: drying and wrinkling of the anterior
chamber of the eye and cornea
● Putrefaction: caused by invasion of the intestinal
ECCHYMOSES microorganisms of the person who died
(Bruise or Pasa) ○ Normally, there is intestinal normal flora
﹘ Normal flora is helpful to our lives but after
we died, normal flora will invade our body to
Brought about a trauma helps our body putrefy (nabubulok)
introduced to a body ○ Manifested by the greenish discoloration of the
abdomen
● Autolysis:
○ Self-digestion of cells
○ Auto: means self
○ This is due to the rupture of lysosomes within the
cell
○ Brought about by lysosomes inside the cell
- Lysosomes: “Suicide sac of the cell”
C. Autopsy
● All autopsies require authorization
● Prosector:
Opposite of postmortem lividity, ○ May be a pathologist or a pathology resident
does not disappear purplish on ○ In-charge of the actual dissection of the body
pressure ● Authorization should come from the next-of-kin of
the deceased in the following order (hierarchy):
1. Legal spouse
2. Adult children (18 years old and above)
On incision, No oozing of blood
3. Parents
4. Adult siblings (18 years old and above)
5. Guardians
6. Responsible party of the state: need of
notarized consent if wala talagang kumukuha ng
bangkay

AT | DP | ST | MS | CP | PP | RC | CM 5
 - Filtration method
Note: A healthy, relaxed, sedentary 70 kg man who is killed instantly
in an accident will usually have organ weights in these ranges: - Plasma-thrombin method
● Right lung: 300-400 gm - Carbowax method
● Left lung: 250-350 gm b. Smears
● Heart: 250-300 gm ● Avoid drying of smears prior to fixation unlike
● Liver: 1100-1600 gm (1.1 - 1.6kg) blood smears
● Adrenals: 4 gm or so each ○ Blood smears: smears needs to be dry prior
● Thyroid: 10-50 gm immersing in the ethanol
● Spleen: 60-300 gm
○ Smears of exfoliative cytology: this must be
● Brain: 1150-1450 gm (1.2 - 1.5kg)
fix (undergo fixation) while they are still
moist
V. BASIC DIAGNOSTIC CYTOLOGY ● Fixatives:
● Cytology: study of cells ○ Fix while it is moist
● Microscopic examination of cells from different body ○ Equal parts of 95% ethanol and ether
sites ﹘ Used in the Pap’s smear
● Divisions: ﹘ Best kinds of fixatives for smear
○ Exfoliative cytology (Pap’s Smear) specimens however it is not commonly
﹘ Pap’s staining method: staining method of used
choice for exfoliative cytology ○ 95% ethanol
○ Fine Needle Aspiration (FNA) - Commonly used in lab
○ Remember if gonna put the fixatives in the
Exfoliative Cytology container:
● Branch of general cytology ﹘ Spray fixative: slide should be kept at a
● Microscopic study of cells that have been distance of 12 inches or 1 foot away
desquamated from epithelial surfaces from that spray fixatives container
● Usually recommended for: Precautions during fixation:
○ For assessing malignant or cancerous conditions 1. Identify the slides before preparing smears
(detection of malignant cells) 2. Use paper clips to the identified end of the slide before
○ For detection of asymptomatic cancer in women preparing smears
(*Detection of precancerous cervical lesions 3. Smears should be placed into the fixative container
in women) immediately after preparations
○ *For assessment of female hormonal activity 4. Place each smear in fixative by a single uninterrupted
in case of sterility and endocrine disorders motion to avoid rippling of smeared material
○ Determination of genetic sex 5. Avoid striking the bottom of the fixative container forcefully
○ Determination of the presence of possible to prevent dislodging the cells
infection (Detection of infectious agents)
*2 Bold: major topics about exfoliative cytology NON-GYNECOLOGIC SPECIMENS
● Example: this is where we use Pap’s Smear (this is ● These specimens which are other than Pap’s smear
where we see Trichomonas Vaginalis). Trichomonas Respiratory Tract Specimens:
vaginalis is an infectious agent. This means that 1. Sputum:
Exfoliative Cytology can be used for that purpose ● Obtain at least 3 consecutive morning sputum
because we can see infectious agents in the Pap’s specimens (through deep cough)
smear. ● Use wide-mouthed jar with Saccomno’s fixative
● Specimens for examination: ● Sputum induction: inhalation of aerosol solution for
○ Vaginal smears 20 minutes
○ Endometrial and endocervical smears ● Alveolar macrophages: Sputum from deep cough
○ Prostatic and breast secretions (able to know if it is really sputum it must find alveolar
○ Gastric or bronchial secretions macrophages in it)
○ Pleural and peritoneal fluids ○ Alveolar macrophages A.k.a “Dust cells”
○ Sputum ● Saliva: Absence of alveolar macrophages
○ Smears of Urine Sediment 2. Bronchoalveolar lavage/bronchial washing (BAL)
○ Cerebrospinal Fluid ● Performed in patients with AIDS in order to rule out
Pneumocystis carinii
● Fixation ○ New name: pneumocystis jiroveci
a. Fluid specimen: the one put in the bottles that 3. Bronchial brushings
received in the histopathology ● Specimen is directly smeared onto 2 labeled slides
● Fixatives: by pull technique
○ 50% alcohol (all types of effusion) ● Fix the slides immediately
○ Saccomano’s fixative (50% ethanol and 2% ● Failure to fix the slides within a few seconds will
carbowax) produce air drying artifacts
● Centrifugation: 4. Peritoneal, Pleural, & Pericardial fluids:
○ 2000 rpm for 2 mins ● These are effusions coming from close cavities of the
○ Supernatant → decanted body since they are effusions they will generally form
○ Sediment → smeared clots
- directly to glass slide ● To prevent jelly-like clots, add 300 units of Heparin
- cytospin on slides with egg albumin per 100 mL of the aspirate
○ Extra sediment → cell block technique
AT | DP | ST | MS | CP | PP | RC | CM 6
 ● Methods of collection are: paracentesis, ○ Not permeable to stain
pleurocentesis/thoracentesis and pericardiocentesis 3. Adhesive agents (used for cytology)
○ Peritoneal fluid: comes from the abdominal ● Pooled human serum or plasma
area ● Celloidin ether alcohol
﹘ Paracentesis: a procedure that removes ● Leuconostoc culture
peritoneal fluid from the abdomen through a
slender needle GYNECOLOGIC SPECIMENS: PAP SMEARS
○ Pleural fluid: comes from the lung area ● Developed by George Papanicolau: Father of
﹘ Pleurocentesis or Thoracentesis: medical exfoliative cytology
procedure used to remove excess fluid from ● Screening tool; NOT DIAGNOSTIC
the pleural space and diagnose or treat
pleural effusions Three anatomic sites from where Pap’s smear is derived:
○ Thoracentesis is minimally invasive, 1. Upper lateral 3rd of the vaginal wall
doctors may also use it as a palliative 2. Ectocervix
treatment for certain pleural 3. Endocervix
mesothelioma patients
○ Pericardial fluid: comes from the heart area Remember where the zone of cancer cells arise:
﹘ Pericardiocentesis: a procedure performed ● The following are important for the diagnosis of
to remove pericardial fluid from the female genital cancer
pericardial sac ● Area between the ectocervix and endocervix is T
5. Gastrointestinal specimens: zone or transformation zone → cancer cells are seen
● Collection is usually done to exclude the possibility of here
malignant tumors ● The upper lateral 3rd of the vaginal wall is for the
● Type of specimen: evaluation of female hormonal status/cytology
○ Gastric lavage ● If the specimen is derived from the 3, it is for the
○ Gastric brush detection of female genital cancer
○ FNA (for submucosal lesions)
6. Urine/Urinary Tract Specimens/ Urinary Tract
Cytology:
● Major goal: diagnosis of urothelial malignancy
● Prostatic carcinomas are rarely found in urinary
specimens
● At least 50mL of sample is needed
● First voided urine should be discarded (overnight
exposure cause degeneration of cells)
○ Too acidic and cause degradation of cells
● Second urine is preferred
● Use of preservatives is not recommended
● Urine is one of the many kinds of specimens that ● Ectocervix: yellow circle on the cervix part
would require adhesive agents
● Types of specimens:
○ For males: Voided urine
﹘ Voided urine: it is a kind of specimen that
comes out from urethra naturally; there is a
● Endocervix: 2 green small circles
possibility that vulvar cells are included
● Upper lateral 3rd of the vaginal wall: wall of the
○ For Females: Catheterized specimen
vagina
﹘ to prevent contamination with vulvar cells
﹘ It is inevitable that vulvar cell will go with the
urine as it pass through the vulva
○ Washing from bladder or renal pelvis

Adhesion:
1. Specimens requiring addition of an adhesive agent:
● Urinary sediment: very watery
● Bronchial lavage
● Specimen that utilizes proteolytic enzymes during
processing. Examples:
○ Trypsin
○ Concentrated sputum and enzymatic lavage ● Speculum and cytobrush: duck like device + brush
specimen from gastrointestinal tract
2. Characteristic of adhesive agents: Vaginal Hormonal Cytology
● It must be permeable to both fixative and stain ● Relatively inexpensive
● It must not retain the stain ● May be performed regularly even in pregnant women
● Egg albumin: not recommended as an adhesive without undue risk
agent only in cytology, but may be used in paraffin ● Vaginal smears for such purpose are taken from the
embedded cells or sections upper lateral third of the vaginal wall
○ Retain the stain ● **CHMI (Cytohormonal Maturation Index)
AT | DP | ST | MS | CP | PP | RC | CM 7
 ○ Quantitative evaluation done under vaginal
hormonal cytology
○ General steps:
- Count 100 cervico-vaginal cells
- Classify cells as: parabasal, intermediate,
and mature superficial cells
- Report the findings in the following manner:
P/I/S
- Observe for the following:
➔ Shift to the left: increased number of
parabasal cells
◆ Example: 100 / 0 / 0
➔ Shift to the right: increased number of
mature superficial cells
◆ Example: 0 / 0 / 100
➔ Shift to the midzone: increased
number of intermediate cells
◆ Example: 5 / 90 / 5
Precautions observed during vaginal smear preparation:
● The patient should not have been douched or undergone vaginal
examination for at least 24-48 hours before smears are prepared
● The glass pipette used should be absolutely dry since presence
of water will distort cellular details
● No lubricant or powder should be used on the examiner’s gloves
● Smears should be spread thinly in a rotary motion instead of by
pull-apart method
● All materials should have been ready before smear collection is
done
● Scraping from the lateral vaginal wall with wooden spatula is
recommended only for hormonal studies
● For detection of female genital cancer, combined vaginal and
cervical smear is the method of choice
CELLS FOUND IN CERVICO-VAGINAL SMEARS
1. Mature superficial cells/superficial cells
● With dark pyknotic nuclei
● With “true acidophilia” (under estrogen influence)
● Pseudoacidophilia: “false acidophilia” Observed
due to:
○ Drying of smears especially before fixation
○ Prolapse and drying of vaginal epithelium
○ Infection
○ Chemicals
2. Intermediate cells
● Medium-sized
● Polyhedral or elongated cells
● Basophilic cytoplasm showing vacuoles
○ Blue cytoplasm
● Navicular cells:
○ Boat-shaped intermediate cells
○ Folds/curls on edges (difference with pregnancy
cells)
● Pregnancy cells:
○ Round, oval, or boat-shaped cells
○ With translucent basophilic cytoplasm
○ Nucleus pushed aside or towards the cell
membrane
○ With double-walled boundary appearance
(deeper blue stain of the cytoplasm at the
periphery)
3. Parabasal cells
● Thick, round to oval
● Smaller than intermediate cells
● Similar to fried fresh eggs with sunny-side up
○ Group into 2-3 cells
● Normally found from two weeks of age to puberty,
after childbirth, abortions, and after menopause
4. Endometrial cells
● Similar in appearance to parabasal cells
● Slightly cylindrical with less basophilic cytoplasm
● Occurring in groups of 3 or more cells
● Found during and 1-10 days after menstruation
5. Endocervical glandular cells
● Cytoplasm is usually stained pale blue/gray, finely
vacuolated
● Nuclei with finely granular chromatin
● Having a “honeycomb” appearance when viewed on
end
6. Basal cells
● Small, round to slightly oval cells
● With relatively large nuclei (occupying more than half
of the cell)
● Strongly basophilic cytoplasm
● Found before puberty and after menopause
7. Doderlein bacillus
● Gram-positive, slender rod shaped
● A.k.a Lactobacillus acidophilus
○ Loves the environment of the vagina
● Most common organism of the normal vaginal flora
● Numerous naked nuclei with many Doderlein bacilli:
○ Last trimester of pregnancy
○ Diabetes mellitus
○ Infection
○ Estrogen deficiency
8. Candida albicans
● Yeast
● Candidiasis commonly seen in:
○ Diabetic patients
○ Patients taking oral contraceptives
○ Immunocompromised states
○ Leukemia
○ Lymphoma
9. Trichomonas vaginalis
● Pear-shaped (discolored upon the addition of Pap
stain)
● Blue-green to blue-gray color in pap smear
● Causes strawberry cervix
10. Gardnerella vaginalis
● Coccobacilli
● Clue cells: squamous epithelial cells filled with
coccobacilli (Gardnerella vaginalis)
11. Koilocytes
● Squamous epithelial cells that shows the cytopathic
effects of HPV
● Cell with an atypical “wrinkled prune” nucleus
surrounded by a perinuclear halo
● Presence of koilocytosis is diagnosed as low-grade
squamous intraepithelial lesion (Bethesda system)
What is Ferning?
● Cervical mucus exhibits a “palm leaf” pattern
● Due to formation of salt crystals
● Signifies a high, persistent estrogen effect
● One of the basis of the diagnosis of early pregnancy
HISTOPATHOLOGIC TECHNIQUES
I. QUALITY ASSURANCE AND DOCUMENTATION
1. Histopath reports
● Surgical pathology
● Cytopathology report
● Autopsy report
○ 3 copies prepared per report delivered:
- One to the patient
- One to the physician
AT | DP | ST | MS | CP | PP | RC | CM 8
 - One to the file of the laboratory ● Demonstration of soluble substances such as lipids
2. Signatories: only pathologist signs in histopath lab, med and carbohydrates
techs do not, only in other sections of the lab ● Immunofluorescence and immunochemical staining
● Request forms: signed by the patient’s physician ● Some specialized silver stains, particularly in
● Result forms: signed only by the pathologist, not the neuropathology
medtechs
○ RMT’s are only required to sign result forms from Two methods of Preparing Frozen Sections:
other section of the laboratory but not in histopath ● Cold knife Procedure:
3. Specimen handling ○ Almost any microtome can be used
● FIX FIRST! ○ Uses carbon dioxide
○ Prior to labeling the jar ● Cryostat Procedure (cold Microtome)
● Label ○ Optimum working temperature: -18 to -20℃
○ Also important but fixation must be done first ○ Cryostat: a refrigerated cabinet in which a
4. Routine Turn-over of Results modified microtome is housed
● Surgical pathology and cytology: within 24 hours ● All the controls to the microtome are operated from
● Frozen Section: 5-15 minutes outside the cabinet
○ Why? The patient is still undergoing operation ● Presently, the rotary microtome is the type of choice
and need the result immediately for the physician ● Mounting media for cryostat sections:
to decide ○ Water
● Autopsy report: 7 days ○ 20-30% bovine albumin
5. Storage of Specimen, Tissue Blocks & Slides ○ Von apathy’s gum syrup
● Specimen: 1 month to 1 year ○ O.C.T: best! synthetic water-soluble glycols and
○ For solid organs like: resins
- Lungs
- Kidneys Commonly used methods of freezing:
- Amputated foot, etc. ● Liquid nitrogen
○ They have long storage time because they are ○ Most rapid of the common freezing agent
irreplaceable and only have one specimen ● Isopentane cooled by liquid nitrogen
○ May kakailanganin pa yung pathologist sa ● CO2 gas
specimen ● Aerosol sprays
● Tissue Block: 3-10 years
● Slides: Indefinitely Staining methods for Frozen Sections:
○ But depends on the finding on that slide ● Hematoxylin and Eosin
○ This type of H&E is a Progressive type
II. FRESH TISSUE EXAMINATION ○ While the other type of H & E (not this part) is
Note: sa fresh tissue examination, hindi gumagamit ng fixative Regressive
Methods: ● Thionine
1. Teasing/Dissociation ● Polychrome Methylene Blue
● Fresh tissue (without fixative) → placed on a watch ● Alcoholic Pinacyanol Method
glass with isotonic solution → view cells using Phase
Contrast/Brightfield Microscope III. PROCESSING FOR PRESERVED TISSUE EXAMINATION
2. Crushing/Squash Preparation Mnemonic: “F.D.C.I.E.T.S.S.M.L”
● Tissues (<1 mm in thickness) → Placed between 2 ● Fixation
slides → a vital stain is applied → slightly press the ● Dehydration
two slides ● Clearing
● Vital stain: stains living cells Squashing or crush prep ● Impregnation
3. Smear Preparation: cellular materials are spread lightly ● Embedding
over a slide by means of a wire loop,applicator ● Trimming
stick/another slide ● Sectioning
Smear Preparation Techniques: ● Staining
● Streaking ● Mounting
● Spreading ● Labeling
● Pull-apart
● Touch preparation FIXATION
○ a.k.a : Impression smear ● Preserving fresh tissue for examination
○ You cut the organ and the surface is allowed to ● First and most critical step in histotechnology
touch the slide (ididikit molang sa slide yung free ● Ideal time to perform fixation is within 20-30 minutes
surface ng tissue) after interruption of blood supply
4. Frozen Section: normally used when a rapid diagnosis of ● Primary aim: (major goal of preservation) to preserve
tissue is required the morphologic and chemical integrity of the cell in
○ Introduced by Julius Cohnheim as life-like manner as possible (like taking an actual
Application: photograph of the cell)
● Rapid pathologic diagnosis during surgery ● Secondary aim: to harden and protect the tissue
○ Still in the operating room doing the surgery from the trauma of further handling
○ Waiting for the diagnosis for about 5-15 mins. ○ Human organs are naturally soft
Dapat may resulta na ○ Easily crumble and fall apart when processing
● Enzyme histochemistry ○ Example : human brain (spongelike kasi siya)
AT | DP | ST | MS | CP | PP | RC | CM 9
 ● Fixatives have the property of forming cross-links
between proteins
● Stabilization of proteins (that make up the
organ/tissue): Most important reaction for
maintaining tissue morphology
● Correct fixative-to-tissue ratio: 20:1 (also in
decalcifying)
● Usual fixation temperature (surgical specimens):
Room Temperature
○ 24 hours at room temperature
○ You may speed up by putting it in a warmer
temperature but it is not advisable
Practical Considerations of Fixation:
● Speed:
○ Specimen must be placed in the fixative as soon
as it is removed from the body
● Rate of Penetration of Formalin:
○ 1mm per hour
Differences between Formalin and Formaldehyde?
ANSWER: None. They are just the same. However, before, formalin
is considered as a brand name of formaldehyde
● Volume:
○ Ratio between fixative and tissue is 20:1
○ 20x the tissue volume
● Duration of Fixation:
○ 24 hours for routine fixation of surgical specimen
Two mechanisms involved in Fixation:
● Additive fixation: whereby the chemical constituent
of the fixative is taken in and becomes part of the
tissue. Example:
○ Formalin
○ Mercury
○ Osmium tetroxide
● Non-additive fixation: whereby the fixing agent is
not taken in, but changes the tissue composition and
stabilizes the tissue by removing the bound water
attached to hydrogen bonds of certain groups within
the protein molecule. Example:
○ Alcoholic fixatives
- Alcohol is frequently utilized as dehydrating
agents. Wherein, in the dehydration
process, they preserve the tissue itself
Main factors involved in fixation:
● Hydrogen ion concentration (pH)
○ Satisfactory fixation: pH 6-8
● Temperature
○ Surgical specimen: room temperature
○ Electron Microscopy and Histochem: 0.4℃
● Thickness of section: tissue blocks should be either
small or as prescribed by the tissue processor
manufacturer (as there are manufacturer that requires
thinner tissue for their machines)
○ 1 to 2 mm2 for electron Microscopy
○ 2 cm2 for light microscopy→ thin (no >0.4 cm for
light microscopy) or as prescribed by tissue
processor manufacturer
○ Brain: usually suspended whole in 10% buffered
formalin from 2 to 3 weeks
- Similar to sponge in terms of softness
- Crumble apart in the microtome process
● Osmolality
● Concentration
● Duration of Fixation
Notes
● One of the goals of fixation is to harden the tissue
● Brain
○ Hindi muna cinu-cut prior to fixation. Unlike other organs na
icucut muna tapos ilalagay sa fixative
○ Buo munang ifi-fix sa formalin. WHY? Kasi nga masyadong
malambot yung human brain. Need na mapatigas
○ Must be suspended in formalin (whole) for about 2-3 weeks
to have a prior penetration ng formalin sa buong organ
○ Remember:
- The formalin penetrates at a rate of 1mm/hr (super
bagal kaya dapat matagal din naka submerge yung
brain sa formalin para properly penetrated siya)
TYPES OF FIXATIVES
1. According to Composition
a. Simple fixative
● One component
● Example: Glacial acetic acid
○ Causes the tissue to swell
○ Solidifies at 17 deg C: glacial acetic acid
○ Destroys mitochondria and golgi bodies
○ Fixes the nuclei protein
○ Can be used alone but commonly we use it
together with other fixatives kasi pwede
magkaroon ng disadvantages sa tissue
b. Compound Fixative
● Made of 2 or more component fixatives
● Used to neutralize the effect of simple fixatives
against each other (to minimize its adverse
effects)
● Example: Zenker’s Fluid which contains:
○ Glacial acetic acid: causes the tissues to
swell
○ Mercuric chloride: causes the tissue to
shrink
Notes:
● Compound fixatives are made for preventing yung mga hindi
kanais nais na effect ng simple fixatives if they are used alone
● For example:
○ Kapag ginamit mo lang ay glacial acetic acid, it is possible
that tissues will get swollen → preserved nga but swollen
naman
○ If mercuric chloride alone, it is possible that the tissues will
shrink → preserved nga pero shrunken naman
● Zenker’s Fluid:
○ Glacial acetic acid + Mercuric chloride = they will neutralize
the effects of each other
- The glacial acetic acid will neutralize the shrinking
effect of mercuric chloride
- The mercuric chloride will neutralize the swelling effect
of glacial acetic acid
2. According to Action
a. Microanatomical: for general microscopic study of
tissue structures. Can preserve almost all parts of the
tissue or cells (pero yung iba sakanila can preserve
the nucleus or the cytoplasm better) Example:
● 10% Formol Saline
● 10% BNF
● Heidenhain’s Susa
● Bouin’s
● Formol sublimate
● Zenker’s solution
● Zenker-Formol
● Brasil’s
AT | DP | ST | MS | CP | PP | RC | CM 10
 b. Cytological Fixatives: specific parts and particular
- Paraformaldehyde: white precipitates
microscopic elements of the cell itself. Preserve a that may form after prolonged standing
particular part of the cell that is better than the others. especially at low temperature
Three kinds: ➔ Removed by: (F10):
1. Nuclear Fixatives: ➢ Filtration
● Preserves the nuclear structures of the cell ➢ Addition of 10% methanol
● Contain glacial acetic acid which preserves ○ Acid formaldehyde hematin:
the nucleoproteins - Brown or black granules that may
obscure microscopic details
● General pH: 4.6 or less
- Can’t see details or abnormalities
● Example: (FC si New Boui Heiden) beneath these granules
○ Fleming’s - Removed by: (KLSA)
○ Carnoy’s ➔ Kardasewich method
○ Bouin’s ➔ Lillie’s method
○ Newcomer’s ➔ Saturated alcoholic picric
○ Heidenhain’s Susa acid
➔ Alcoholic potassium
hydroxide (KOH)
2. Simple Fixative
a. Cytoplasmic Fixatives: for preserving the Note:
cytoplasmic structures ● Formaldehyde = Formalin
● Must never contain glacial acetic acid ● Formalin is a brand
→ destroys the mitochondria and golgi
bodies (remember that these structures 2. 10% Formol-saline
are seen in the cytoplasm. Thus, you ● Recommended for CNS and general post mortem tissues
won't be able to preserve the
cytoplasmic structures) 3. 10% BNF (Buffered Neutral Formalin)
● General pH: greater than 4.6 ● Best fixative for tissues containing iron pigments an for
● Examples: (F2HOR) elastic fibers (past board exam question)
● Best general tissue fixative
○ Fleming’s fluid without acetic acid
○ Helly’s Fluid 4. Formol-Corrosive (Formol-sublimate)
○ Formalin with post-chroming ● Recommended for routine post-mortem tissues
○ Regaud’s (Moller’s)
○ Orth’s 5. Glutaraldehyde
● Enzyme histochemistry and electron microscopy (past
b. Histochemical Fixatives: board exam question)
● Preserves chemical constituents of the ● 2 concentration:
○ 2.5% solution: small tissue fragments and needle
cell biopsy
● Does not contain glacial acetic acid ○ 4% solution: larger tissue specimens
● Examples: (10EAN)
○ 10% Formol Saline 6. Formol-Calcium
○ Absolute Ethanol/ETOH ● For lipids
○ Acetone
○ Newcomer’s Fluid - function both as 7. Karnovsky’s Paraformaldehyde-glutaraldehyde solution
a nuclear fixative and histochemical ● For electron cytochemistry
fixative 8. Acrolein
● For electron cytochemistry
3. According to Component
Summary:
ALDEHYDE FIXATIVE ● CNS = 10% Formol-Saline
● Iron and Elastic & BEST GENERAL tissue fixative = 10% BNF
1. Formaldehyde or Formalin ● Post-Mortem = 10% Formol-Saline & Formol-Corrosive
● Usual fixation time: 24 hours. (Naka immerse ang ● Enzyme Histochemistry and Electron Microscopy =
specimen) Glutaraldehyde
● Room temperature for surgical specimen and light ● Small tissue fragments an Needle Biopsy = Glutaraldehyde
microscopy 2.5%
● Gas produced from the oxidation of methanol ● Larger tissue fragments = Glutaraldehyde 4%
● Two concentrations ● Lipids = Formol-Calcium
○ 40% formaldehyde: ● Electron Cytochemistry = KPG & Acrolein
- Considered as “stock solution”: not
used for routine fixation as it is high in
concentration and may over-harden
tissues especially the outer part/layer of METALLIC FIXATIVES
the tissue/organ
- Needs to be diluted in order to be Mercuric Chloride (Z2SHOC B-5): most commonly used metallic
appropriate for routine fixation fixative
○ 10% formaldehyde: Some Advantages:
- Considered as “working solution”; ● Routine fixative of choice for tissue photography (past
used for fixation of tissues board exam question)
- Unstable solution as it may form artifacts ● Permits brilliant metachromatic staining of cells
○ Artifacts formed: Some Disadvantages:

AT | DP | ST | MS | CP | PP | RC | CM 11
 ● Causes tissue shrinkage ● One of the disadvantage: Polarization
● Decreases amount of demonstrable glycogen ○ Movement of glycogen granules towards the end or
○ Glycogen is the storage from of glucose in the human poles of the cells
tissue Example:
○ Sometimes, the physician assesses if there is enough 1. Methanol
glycogen stored in the tissues ● Used for fixation of blood smears and bone marrow (BM)
● Corrodes all metal, except for the nickel alloy (Monel) smears
● Produce black granular deposits removed by ● Dangerous, toxic type of chemical and can penetrate the
De-zenkerization technique skin so always wear gloves
○ Removal of the black granular deposits of mercury 2. Ethanol
chloride ● Fixative and dehydrating agent
○ Uses alcoholic iodine ● For fixation of cytologic smears
○ Example: Pap smear: uses 95% ethanol (commonly
Examples: (Z2SHOC B-5) used)
1. Zenker’s Fluid (with glacial acetic acid) 3. Carnoy’s fluid
● Small pieces of liver, spleen, connective tissue fibers and ● Most rapid fixative (fixation time: 1-3 hours)
nuclei ● Used for preservation of chromosomes (past board exam
2. Zenker-Formol (Helly’s Solution) question)
● Pituitary gland, bone marrow and blood containing organs ● Used for fixation lymph glands and urgent biopsies
(ex: spleen and liver) ● Used for glycogen fixation
3. Heidenhain’s SuSa ● Used for preservation of brain tissue for rabies diagnosis
● Tumor biopsies especially the skin 4. Alcoholic Formalin (Gendre’s Fixative)
● “SuSa”= from 2 german words: ● Useful in preserving sputum
○ Sublimat “Su”: mercuric chloride 5. Newcomer’s Fluid
○ Saure “Sa”: acid ● Both a fixative for both nuclear and histochemical fixative
4. Schaudinn’s Fluid ● For preservation of mucopolysaccharides and nuclear
5. Ohlmacher’s Fluid proteins
6. Carnoy-Lebrun Fluid
7. B-5 fixative OSMIUM TETROXIDE FIXATIVES
● Commonly used for bone marrow biopsies
● Should be kept in a dark-colored, chemically clean bottle to
Chromate prevent evaporation and reduction by sunlight or organic
● Chromic acid matter
● Potassium dichromate ● Inhibits hematoxylin and makes counterstain difficult
● Regaud’s (Moller’s) ○ Produces black precipitates (osmic oxide)
● Orth’s fluid: ○ Prevention: add saturated aqueous mercuric chloride
○ For Rickettsia and other bacteria ○ Remedy: black osmic oxide crystals may be dissolved
○ For study of early degenerative process in cold water
○ Precaution: may cause conjunctivitis or blindness
Lead Fixative: are generally for acid mucopolysaccharides ● Example:
● Example: umbilical cord/wharton’s jelly ○ Flemming’s Solution
- Nuclear fixative
PICRIC ACID FIXATIVE ○ Flemming’s without acetic acid
- Cytoplasmic fixative
● Used as:
○ Fixative TRICHLOROACETIC ACID
○ Decalcifying agent
○ Stain ● Function not also a fixative but acts as decalcifying agent
● Always in liquid form because it’s highly explosive when
dry
ACETONE
● Will produce excessive yellow staining of tissues
● Picrates are formed upon protein; precipitates are soluble in
water; hence tissues must be first rendered insoluble by ● A fixative but is used at ice cold temperature (-5 to 4℃)
direct immersion in 70% ETOH ● Uses:
● Picrates fixatives must never be washed in water before ○ Preservation of water-diffusible enzymes (such as
dehydration lipases and phosphatases)
● Example: ○ Preservation of Brain tissue for Rabies diagnosis
○ Bouin’s solution
- Recommended for embryos and pituitary HEAT FIXATION
biopsies
○ Brasil’s alcoholic picroformol ● Direct flaming fixation
● Microwave fixation: non chemical technique useful for
GLACIAL ACETIC ACID demonstration of neurochemical substances in brain (such
as acetylcholine)
○ Optimum temperature 45-55℃
● Solidifies at 17℃
○ Floatation water bath: 45-50℃
● Fixes the nucleoproteins
● Causes the tissues to swell
● Destroys the mitochondria and golgi bodies FIXATIVE FOR ELECTRON MICROSCOPY
○ Not part of Cytoplasm Fixative
● Glutaraldehyde
ALCOHOLIC FIXATIVES ● Platinic chloride (PtCl3)
● Platonic chloride-formalin (Zamboni’s fixative)
● Gold Chloride (AuCl)
● Generally recommended for: glycogen fixation
AT | DP | ST | MS | CP | PP | RC | CM 12

● Osmium Tetroxide
● 10% BNF
FIXATIVE FOR ELECTRON MICROSCOPY
● Fixation may be retarded (“to slow down”) by:
○ Size and thickness of the tissue (Too thick/ large)
○ Presence of mucus
○ Presence of blood
○ Presence of fats
○ Cold temperature
● Fixation may be enhanced by:
○ Size and thickness of the tissue
○ Agitation
○ Moderate heat (37-56℃)
DECALCIFICATION
● Done after fixation; Used to soften hard tissues
○ More concentrated acid solutions decalcify bone
more rapidly but are more harmful to the tissue
○ High concentrations and greater amount of fluid
will increase the speed of the process
● The recommended ratio of fluid to tissue volume for
decalcification is 20:1
● Heat will serve to hastens decalcification but it also
increases the damaging effects on tissues
● At 37°C: impaired nuclear staining of Van Gieson's
stain for collagen fibers
● At 55°C: tissue will undergo complete digestion within
24-48 hours
● Optimum temperature: Room temperature at 18-30°C
● The ideal time required for decalcifying tissues is
24-48 hours
● Dense bone tissues usually require up to 14 days or
longer in order to complete the process
Decalcifying agents:
1. Acids
2. Chelating agents
3. Ion exchange resins
4. Electric ionization
Some examples of Acid decalcifying agents:
1. Nitric Acid: Most commonly used decalcifying agent
● Perenyl’s fluid: function as both decalcifying
agents and tissue softener
● Phloroglucinol-Nitric Acid: acts as the fastest or
most rapid decalcifying agent
2. 5 % Formic acid: Best general decalcifying agent
● Both fixative and decalcifying agents
● Formic acid is recommended for small pieces of
bones and teeth
3. Hydrochloric acid
● Von Ebner’s Fluid: recommended for teeth and small
pieces of bones
Extent of Decalcification
1. 3 ways to measure extent of decalcification:
● Physical/Mechanical Test: simplest but inaccurate
due to subjectivity
○ Done by touching or bending tissue with the
fingers
○ Decalcified tissues:
- Commonly have diminished consistency and
are softer to touch
○ Alternate method:
- Pricking the tissue with a fine needle or a
probe
● X-ray or Radiological Method: most ideal, most
sensitive, most reliable & most expensive
● Chemical method (Calcium Oxalate Test): Simple,
reliable, recommended for routine purposes
Tissue Softeners
1. For unduly hard tissues that may damage the microtome
knives
● 4% aqueous phenol
● Molliflex
● 2% HCl
● 1% HCl in 70% alcohol
DEHYDRATION
● Aim: to remove fixative and water from tissue and
replacing them with dehydrating fluid in preparation
for impregnation
● Dehydrating fluids are generally used in increasing
or ascending strength (all the aqueous tissue fluids
are removed but with little disruption to the tissue due
to diffusion currents; to prevent tissue disruption)
Commonly used dehydrating agents:
1. Alcohol: most commonly used dehydrating
● Ethanol:
○ For routine dehydration of tissues
○ Also use as fixative
○ Best dehydrating agent
● Methyl alcohol: employed for blood and tissue films
● Butyl alcohol: utilized in plant and animal
microtechniques
● Industrial methylated spirit (denatured alcohol):
ethanol + small amount of methanol
● Isopropyl alcohol
2. Acetone
● Used as a fixative, in particular to brain tissue for
rabies patients
3. Dioxane (Diethylene dioxide)
4. THF (Tetrahydrofuran)
● Dioxane and THF: both of these fluid functions as
dehydrating agents and clearing agents (universal
solvent)
5. Cellosolve (Ethylene glycol monoethyl ether)
6. Triethyl phosphate
Additives to dehydrating agents:
1. 4% phenol
● 4% phenol + 95% ETOH baths: act as a tissue
softener
2. Anhydrous copper sulfate: both dehydrating agent and
indicators of H2O content (100% ETOH)
● Indicator of H2O content at the last ethanol bath
○ Sa last ethanol bath kasi, wala ng water, or dapat
wala na totally kase nga nag dedehyrate ka. Pero
kapag may natira pang water doon it means you
have an incomplete dehydration
● Normally white in color = complete dehydration
● If the copper sulfate is hydrated: blue in color
○ Hydrated copper sulfate = incomplete
dehydration process
A typical dehydration sequence for specimens NOT more than
4mm thick would be:
1. 70% ethanol: 15min
2. 90% ethanol: 15 min
3. 100% ethanol: 15 min
AT | DP | ST | MS | CP | PP | RC | CM 13
 4. 100% ethanol: 15 min ● Best general tissue fixative: 10% buffered neutral
5. 100% ethanol: 30 min formalin
6. 100 ethanol: 45 min ● Best decalcifying agent: 5% formic acid
Note: ● Best dehydrating agent: Ethanol
Other laboratories start at 60% alcohol and then they go ● Best infiltrating/embedding medium: Paraffin wax
higher. And some laboratories start at 70%. The most
important is to use an ascending grade of alcohol, it doesn’t Substitutes for Paraffin Wax
matter as long as you use the ascending grade of alcohol 1. Paraplast
proposed by the laboratory. Hindi pwedeng mag start agad sa ● MP: 56-57℃
100% ● Mixture of highly purified paraffin and synthetic
plastic polymers
CLEARING ● More elastic and resilient than paraffin
● A.k.a Dealcoholization ● For large dense tissue blocks such as bones and
● Process of replacing the dehydrating fluid with a fluid brain
that is miscible with both the dehydrating fluid and 2. Embeddol
the impregnating/embedding medium ● Melting Point: 56-58℃
● They makes the tissue clearer and remove the ● Less brittle and less compressible than paraplast
dehydrating agent 3. Bioloid
● Recommended for embedding eyes
Clearing agents suitable for routine use: 4. Tissue Mat
1. Xylene/xylol ● A product of paraffin, containing rubber, with the
● Most commonly used clearing agent same property as paraplast
● Most rapid clearing agent 5. Ester Wax
2. Toluene ● MP: 46-48 deg C
3. Chloroform ● Harder than paraffin
● Toxic to the liver after prolonged inhalation ● Not soluble in water
● It does not make the tissues transparent ● Soluble in alcohol specifically to 95% ETOH and other
4. Methyl benzoate and methyl salicylate clearing agents
5. Cedarwood oil and clove oil ● Can be used for impregnation even without prior
● Especially recommended for CNS tissues and clearing of the tissue
cytological studies, particularly of smooth muscles 6. Water-soluble waxes
and skin ● MP: 38-42 deg C or 45-56 deg C
6. Citrus fruits oils ● Mostly polyethylene glycols
7. Trichloroethane and petrol ● Most commonly used: carbowax
8. Benzene ○ Carbowax: soluble and miscible with water
● May cause secondary Aplastic anemia (hence does not require dehydration and
9. Aniline oil clearing of the tissue)
● Rather recommended for clearing embryos, insects ○ Suitable for many enzyme histochemical studies
and very delicate specimens
10. Carbon tetrachloride Celloidin (collodion)
● Purified form of nitrocellulose
IMPREGNATION A.K.A INFILTRATION ● May also be used as impregnating medium like
● Process of replacing the clearing agent with the bioloid because it uses in processing of eye
infiltrating medium specimens
● The medium used to infiltrate the tissue is usually the ● Suitable for specimens with large hollow cavities,
same medium used for embedding hard and dense tissues (bones and teeth), large
● 25x the tissue volume tissue sections of the whole embryo
● Four types of tissue impregnation and embedding ● Two methods for celloidin impregnation:
media: 1. Wet Celloidin: recommended for bones, teeth,
○ Paraffin wax large brain sections and whole organs
○ Gelatin 2. Dry Celloidin: preferred for processing of whole
○ Celloidin (Colloidin) eye sections
○ Plastic/Resin Gelatin
● Gelatin and carbowax are slightly similar to one
Paraffin another
● The man who introduced paraffin wax embedding: ○ Carbowax is water soluble, can be used even
Butschlii without dehydration
● Simplest, most common and the BEST ○ Gelatin is also a water soluble thus, dehydration
infiltrating/embedding medium is not also necessary
● Not recommended for fatty tissues ● Rarely used except when dehydration is to be
○ Frozen sections are most suitable for this avoided
● Temperature of paraffin oven: 55-60 deg C ● Used when tissues are for histochem and enzyme
○ Paraffin oven must be maintained at a studies
temperature 2-5℃ above the melting point of ● Impregnating medium for delicate specimens and
the paraffin wax frozen sections because it prevents fragmentation of
Recall: tough and friable tissues when frozen sections are cut

AT | DP | ST | MS | CP | PP | RC | CM 14
Plastic/Resin ● Most common type used today especially for
● Used for electron microscopic studies paraffin-embedded tissues
● Classified into:
○ Epoxy 3. Sliding Microtome
- Bisphenol A (commercial name: Araldite) ● Inventor: Adams in 1789
- Glycerol (commercial name: Epon) ● Most dangerous type due to movable exposed knife
➔ Epon is also utilizes in (base-sledge)
electron microscopy ● 2 types:
- Cyclohexene dioxide (commercial name: ○ Base-Sledge:
Spurr) - For all forms of media
○ Polyester - Block holder: moving
○ Acrylic - Knife: stationary
○ Standard Sliding Microtome:
Recal: - Block: stationary
1. What is Epon? - Knife: moving
Answer: Epon is a plastic impregnating media
4. Rotary Rocking Microtome
2. Fixative for Electron Microscopic study? 5. Vibratome
Answer: Glutaraldehyde ● Used for unfixed, unfrozen specimen sectioning for
enzyme demonstrations
EMBEDDING ● Disadvantage: sections are liable to disintegrate
● A.k.a Casting/Blocking
● Process by which the impregnated tissue is placed 6. Ultrathin Microtome
into a precisely arranged position in a mold ● For cutting sections for electron microscopy
containing a medium which is then allowed to solidify ● Uses diamond knives or broken plate glass
○ Remove tissue from cassette ● Specimen is small, fixed in osmium tetroxide,
○ Fill mold with wax and orientate tissue embedded in plastic
- Nowadays, molds are made of metal. Back
then, paper or plastic were used 7. Freezing Microtome
● Orientation: process by which a tissue is arranged ● Inventor: Queckett in 1847
in precise positions in the mold during embedding,
on the microtome before cutting, and on the slide
MICROTOME KNIVES
before staining
● Temperature of melted paraffin used for embedding:
Microtome Knives Usual Length Description
5-10 C above its melting point of wax
● To solidify embedded tissue: cooled rapidly in a
Plane concave 25mm One side: flat
refrigerator (-5 C) or immersed in cold water
Other side: concave
● The surface of the section to be cut should be placed
parallel to the bottom of the mold in which it is Biconcave 120mm Both sides: concave
oriented
Plane wedge 100mm Both sides: straight
TRIMMING
● Process of removing excess wax after embedding ● Clearance angle: 0-15 degrees
● Excess wax is cut off from the block to expose the ○ How far the knife is away from the tissue block
tissue surface in preparation for actual cutting ● Bevel angle: 27-32 degrees
○ Trimming allows easier cutting for the microtome ● Stages of Knife Sharpening:
● Knife/blade may be used a. Honing: Removes nicks
● Correct shape of the block: four-sided prism or ● Heel to toe direction
truncated pyramid ● Types of hones:
○ Arkansas
SECTIONING ○ Fine carborundum
● A.k.a Cutting or Microtomy ○ Belgium Yellow: gives best result
● The process by which a processed tissue is cut into b. Stropping: Removes burrs
uniformly thin slices (sections/ribbons) to facilitate ● Toe to heel direction
studies under the microscope ● Instrument used:
● 4-6u: Routine tissue microtomy ○ Paddle stop (made of horse leather)
● 10-15u: Frozen section
● 0.5u: Electron microscopic study ● Temperature of Flotation water bath:
○ 45-50℃ (approximately 6-10℃ lower than
Kinds of Microtomes melting point of wax)
1. Rocking Microtome (Cambridge Rocking Microtome)
● Inventor: Paldwell Trefall in 1881 ● Adhesive agents:
● Simplest among the microtomes 1. Mayer’s Egg Albumin
● Disadvantage: difficulty in reorienting the block ● Most common adhesive
● Not recommended for cytology smears
2. Rotary/Minot Microtome ● Contains:
● Inventor: Minot in 1885-1886 ○ Egg white or “Albumen”
AT | DP | ST | MS | CP | PP | RC | CM 15
 - AlbumEn: refers to the egg white b. Auxochromes: responsible for dyeing
- AlbumIn: protein property
○ Glycerol/Glycerin ● Cationic auxochrome: amino group
○ Crystals of thymol: to prevent growth of ● Anionic auxochrome: hydroxyl and
molds carboxyl Groups
2. Dried Albumin c. Dye modifiers (attached on benzene ring)
3. Gelatin ● Ethyl groups
4. Gelatin-formaldehyde mixture ● Methyl groups
5. Starch paste ● Sulphonic Acid
6. Plasma d. Dye-to-tissue mechanisms: tissues will
7. Poly-L-Lysine bind dyes by one of the following
8. 3-APES (3-aminopropyltriethoxysilane) mechanisms
● APES-coated slides: very useful in 1. Electrostatic: majority of tissue-dye
cytology, particularly for cytospin reactions
preparations of proteinaceous or ● Ex: neutral red and light green
bloody material 2. Hydrogen bonding
● Ex: congo red, carmine, weigert-
Recall: type resorcinol dye
● Impregnation: 2-5 deg above MP 3. Van der Waals Forces
● Embedding: 5-10 deg above MP ● Ex: alum hematoxylin solutions
● Floatation water bath: 6-10 deg below MP 4. Physical Staining
● Example: sudan dyes
STAINING ● Sudanophilia: property of tissues
● Natural dyes: obtained from plants and animals to be stained with fat or oil-soluble
1. Hematoxylin dyes, regardless of the type of dye
● Derived from the heartwood of the mexican used, due to their essential lipid
tree (Hematoxylin campechianum) nature
○ Once you have taken hematoxylin on 5. Natural Affinity
this tree, it needs to undergo a particular ● Example: Janus Green
process before you can utilize it
(ripening/oxidation) Methods of Staining:
● Ripening/Oxidation: get the active coloring A. According to the presence of mordant:
form which termed as HEMATEIN 1. Direct Staining
○ Although it can give color, it cannot ● Gives color to the sections by using simple
readily attach to the tissue aqueous or alcoholic dye solutions
○ Mordant: “bridge” that is necessary for ● Mordant is not required
hematein to attach to the tissue ● Example: Methylene Blue
○ Resultant hematein-mordant-tissue 2. Indirect Staining
complex: Dye lake ● The action of the dye is intensified by using a
● Considered as indirect stain (requires mordant
mordant to attach in the tissues) ○ Mordant: bridge or link between the dye and
2. Cochineal dyes tissue
● Extracted from the female cochineal bug ● Example: Hematoxylin (dye lake: mordant-tissue
(Coccus cacti) complex)
3. Orcein
4. Saffron B. According to the presence of a differentiator
1. Progressive Staining
● Synthetic dyes: ● When dye is taken up by the tissue, it is NOT
○ a.k.a. Coal Tar Dyes decolorized (hindi gumagamit ng
○ Derived from benzene and collectively known as decolorization/differentiation)
“aniline dyes” ● Example: H&E
- Benzene ring is not able to give color; ○ Used for frozen sections
chromophore must attach to benzene 2. Regressive staining
to impart color ● Requires differentiator/decolorizer
- Chromogen: benzene + chromophore ● Tissue is first overstrained, then excess stain is
which imparts color temporarily remove/decolorized from unwanted parts of the
- Auxochrome: must attach to tissue
chromogen to produce dye, which ● Example: H&E
imparts color almost permanently ○ Used for routine tissue straining
a. Chromophores: responsible for coloring
property C. According to the resultant color of the tissue
● Quinoid ring 1. Orthochromatic staining
● Azo groups (Congo red) ● Color of the dye is the expected (same) color of
● Xanthene (Eosin) the tissue
● Quinone-imine group ● Color of the dye = color of the tissue
○ Oxazin 2. Metachromatic Staining
○ Thiazins
AT | DP | ST | MS | CP | PP | RC | CM 16
 ● Color of the dye is not the resultant color of the
Cole’s Alcoholic iodine
tissue
● Color of the dye ≠ not color of the tissue
Carazzi Potassium iodate
D. Vital staining: selective staining of living cell constituents
1. Intravital Staining B. Iron Hematoxylin
● Injection of the dye into any part of the animal ● Iron salts are used as oxidizing agents and
body mordant
● Staining while inside the body (gives color inside
Weigert’s ● Ferric chloride
the body) ● In combination with van Gieson’s
● Example: Lithium, Carmine and India Ink stain, can demonstrate connective
2. Supravital Staining tissue elements and Entamoeba
● Staining of living cells immediately after removal histolytica in sections
from the body ○ Van Gieson’s stain: good for
● Staining happens outside the body demonstrating collagen
● Example: ● Standard iron hematoxylin
○ Neutral Red: best vital dye ● For muscles/connective tissue fibers
○ Janus green: for mitochondria
Heidenhain’s ● Ferric ammonium sulfate
○ Nile blue
● For mitochondria, muscle striations,
○ Trypan blue chromatin, and myelin

ROUTINE HEMATOXYLIN AND EOSIN STAINING Verhöeff ● Used for staining elastic fibers
● Most widely used histological stain
● The primary stain is hematoxylin and the Loyez ● Used for staining myelin
secondary stain is eosin

1. Hematoxylin C. Tungsten Hematoxylin

● A natural dye derived from extraction from the ● Mallory’s PTAH (Phosphotungstic Acid
heartwood of the mexican tree (Hematoxylin Hematoxylin)
campechianum) ○ Mordant: Tungsten
● Ripening/Oxidation: may be done by exposing the ○ To ripen: stand in the light for several weeks
substance to air and sunlight (slow) or use potassium for immediate ripening
○ Ripening may be done also by adding oxidizing ○ For staining muscle striations, fibrin and glial
agents such as: fibers
- Hydrogen peroxide
- Mercuric oxide (ripening agent for Harris D. Copper Hematoxylin
hematoxylin) ● Used for study of spermatogenesis
- Potassium permanganate
- Sodium perborate E. Molybdenum Hematoxylin
- Sodium iodate (ripening agent of Ehrlich’s ● Thomas hematoxylin: used for collagen and
hematoxylin) endocrine cell granules
● Waldeyer: first person to use hematoxylin in histology
in 1862 F. Lead Hematoxylin
○ Hematoxylin was originally used as a dye for ● Solcia hematoxylin: used for endocrine cell
clothing granules
● The name of hematoxylin is mordant. Example for
Alum hematoxylin, Aluminum is the mordant. 2. Eosin
● Red acid dye
A. Alum Hematoxylin ● Routinely used in histopathology as a counterstain
● Used in routine H&E after hematoxylin and before methylene blue
● Mordant: Potash alum (potassium aluminum ● 3 forms:
sulfate or simply “alum”) ○ Eosin Y (yellowish): most commonly used
● Produce good nuclear stain ○ Eosin B (bluish): deeper red color
○ Eosin S: Ethyl eosin
Alum Hematoxylins Ripening Agent (Oxidant)
STEPS IN ROUTINE H&E STAINING
Harris Mercuric oxide
Regressive staining:
*Useful general purpose
hematoxylin 1. Xylol (2 changes) for deparaffinization
2. Descending grade of alcohol (rehydration)
Ehrlich’s Natural or sodium iodate 3. Water: removal of pigments is done after rehydration and
right before primary staining
Delafield’s Natural 4. Stain with Harris/Ehrlich’s/Delafield’s
5. Rinse slides in tap water
Gill’s Sodium iodate 6. Acid alcohol (differentiator)
7. Ammonia water (ammonium hydroxide, lithium
Mayer’s Sodium iodate carbonate, Scott’s tap water) for the blueing phase

AT | DP | ST | MS | CP | PP | RC | CM 17
 ●If the blueing agent is not performed, the nucleus will
Victoria Blue ● Used for demonstration of neuroglia in frozen
remain/appear red sections
8. Wash well in running tap water
9. Stain with Eosin Y: secondary staining Lysochromes (Oil ● Not real dyes
● Pale pink: cytoplasm Soluble Dyes) ● They do not have auxochrome groups
10. Ascending grade of alcohol (dehydration) ● They give color to lipids simply because they
11. Xylol/xylene (clearing/dealcoholization) are more soluble in the lipid medium of the
12. Mount then label tissues than in their medium of 70% alcohol
● Examples of oil soluble dyes used for
Question: What will be the expected color of the nucleus if the demonstration of intracellular fats:
○ Sudan Black B: black
MT forgot to use ammonia water?
○ Sudan III: orange
● Red ○ Sudan IV (Scharlach R): red

Results: Periodic ● PAS-positive substances: red/magenta red

● Nuclei: blue to blue black Acid-Schiff ● Mucoproteins are the most common
● Karyosome: dark blue Reaction PAS-positive substances
● Cytoplasm, proteins in edema fluid: pale pink
● Calcium and calcified bone: purplish blue
● Muscle fibers: deep pink MOUNTING
● Refractive index – ratio of speed of light in air and
PAP SMEAR STAINING STEPS speed of light in a specific medium.
1. Fix with 95% ETOH ● Refractive index of glass = 1.518
2. Stain with Harris Hematoxylin A. Resinous Media B. Aqueous Media
3. Acid Alcohol (Refractive Index: greater than or (usually for lipids because resinous
4. Blueing step equal to 1.518) media contain xylene which may
5. Stain with OG-6: stains the cytoplasm of mature dissolve fats)
(superficial cells)
6. 70-95% ETOH: for washing ● Eukitt ● Glycerin: 1.47
7. Stain with EA 36 or 50: stains the cytoplasm of immature ● Entallan ● Gum Arabic (Farrant’s medium):
● DPX: 1.532 1.43
cells (intermediate, parabasal)
● Histomount ● Karo Corn Syrup
8. Dehydrate ● XAM: 1.52 ● Apathy’s medium: 1.52
9. Xylol ● Paramount ● Brun’s Fluid: recommended for
10. Mount and label ● Canada Balsam (Abus mounting frozen sections from
Balsamea): 1.524 water
OTHER STAINS & THEIR USES ● Clarite: 1.544 ● Water: evaporates easily

Benzidine ● Used for staining hemoglobin

IMMUNOHISTOCHEMISTRY
Acridine Orange ● DNA (green fluorescence) INTRODUCTION
● RNA (red fluorescence) ● Immunohistochemistry: use of antibodies as
histological tools for identifying patterns of antigen
Crystal violet ● For staining amyloid in frozen sections and distribution within a tissue or an organism
platelets in blood ● IgG: most commonly used antibody for
immunocytochemistry
Gentian violet ● Formed by the mixture of crystal violet, methyl ● Polyclonal Antibodies
violet, and dextrin ○ Produced by immunizing an animal with an
immunogen that contains the antigen of
Congo Red ● Stain for axis cylinders in embryos
interest
● Used as a 4% aqueous solution in Krajian’s
method of staining elastic tissues, amyloid, ○ Most frequently used animal: rabbit, then
and myelin goat, pig, sheep, horse, guinea pig, others
● Monoclonal Antibodies:
Iodine ● Probably the oldest of all stains ○ Products of an individual clone of plasma
● Stains amyloid, cellulose, starch, carotenes, cells
and glycogen ○ Animal used: mice
● Widely used for removal of mercuric fixative
pigments PREPARING TISSUE FOR IHC
In certain instances, tissue must be prepared as a cryostat
Malachite Green ● Contrast stain for staining ascaris eggs and section and fixed for a few seconds in absolute methanol or
erythrocytes acetone (to preserve immunological activity and prevent
● Used also as a bacterial spore stain
destruction of some of the labile antigenic sites)
1. Proteolytic enzyme digestion
Janus Green B ● Used for demonstrating mitochondria during
intravital staining (or supravital)
● Especially useful for demonstrating:
● Heavy chain immunoglobulins
Night Blue ● Used as a substitute for carbol fuchsin in ○ Complement
acid-fast staining ○ Specific antigens (such as cytokeratin)
● Most commonly used enzymes:
○ Trypsin
AT | DP | ST | MS | CP | PP | RC | CM 18
 ○ Protease
Desmin ● Highly specific for myogenic tumors,
2. Microwave antigen retrieval including leiomyoma (smooth muscle tumor)
● Involves the boiling of formalin-fixed deparaffinized and rhabdomyosarcoma (skeletal muscle
sections in certain solutions such as: tumor)
○ 0.01 M Citrate Buffer (pH 6.0)
○ EDTA at pH 8.0 Glial fibrillary ● Expressed CNS glial cells, particularly
○ Tris-EDTA (pH 9.9 or 10.0) acidic protein astrocytes
3. Microwave and trypsin antigen retrieval (GFAP) ● Most widely used to confirm the diagnosis
4. Pressure cooker antigen retrieval of astrocytoma
● Less time consuming
● Allows for more consistent recovery of many antigens Neurofilament ● Tumors that show neuronal or
(NF) neuroendocrine differentiation will stain
positive for neurofilament (i.e.,
ANTIGENS
neuroblastomas, ganglioneuromas,
EPITHELIAL TUMOR MARKERS neuromas, chemodectomas,
pheochromocytomas)
Keratin ● Highly sensitive marker for epithelial cells,
present in epithelial tumors (carcinoma) S100 Protein ● Low molecular weight calcium-binding
● Also stain positive for keratin: protein that is expressed in CNS glial cells,
mesotheliomas and non-seminomatous Schwann cells, melanocytes, histiocytes,
germ cell tumors chondrocytes, skeletal and cardiac muscle,
● CK7 (Cytokeratin 7: frequently found in myoepithelial cells and some epithelial cells
carcinomas of the lungs, breast, uterus, and of breast, salivary and sweat gland
ovaries (these tumors are typically negative epithelium.
for CK20)
● CK20 (Cytokeratin 20): common in NEUROENDOCRINE MARKERS
carcinomas of colon and stomach (these
tumors are negative for CK7) Neuron- specific ● Provides strong evidence of neural or
● Transitional cell carcinomas of the bladder enolase (NSE) neuroendocrine differentiation
and mucinous ovarian tumors are usually
positive for both CK7 and CK20
Chromogranin ● Recognized as a marker for neuroendocrine
● Renal cell carcinomas, hepatocellular
differentiation
carcinomas, prostatic adenocarcinomas,
thyroid carcinomas and squamous cell
carcinomas (skin, lung and esophagus) are Synaptophysin
usually negative for either CK7 or CK20
GERM CELL TUMOR MARKERS
EMA (Epithelial ● Positive for adenocarcinomas of the breast,
Membrane lungs and kidneys HCG (human ● Synthesized by placental
Antigen) ● Non-reactive for hepatocellular carcinomas, chorionic syncytiotrophoblasts
adrenal carcinomas or embryonal gonadotropin) ● Marker for choriocarcinoma
carcinomas
● Negative for non-epithelial tumors AFP (alpha- ● Used as a marker for endodermal sinus
(sarcomas, lymphomas, melanomas) fetoprotein) tumors showing yolk sac differentiation

CEA (Carcino- ● An oncofetal antigen PLAP ● Used as a marker for germ cell tumors,
embryonic ● Present in carcinomas of the gastrointestinal (placenta-like particularly germinomas
Antigen) tract, pancreas, lung, breast, ovary, uterus alkaline
and cervix phosphatase)
● Especially useful in differentiating between
adenocarcinoma (CEA +) and mesothelioma MESENCHYMAL TUMOR MARKERS
(CEA -)
Myogenic ● Use actin and desmin and/or other muscle
TTF-1 (Thyroid ● Useful in differentiating lung tumors markers such as myo-D1, myoglobin, and
transcription adenocarcinomas from mesotheliomas myogenin
factor-1) ● Positive in thyroid, lung and neuroendocrine
tumors Fibrohistiocytic
tumors
PSA (Prostate ● Useful in the diagnosis of prostatic
Specific adenocarcinoma Vascular tumors ● Factor VII-related antigen, CD31, and Ulex
Antigen) ● Also positive in certain pancreatic and Europaeus I (UEA)
salivary gland tumors
Melanomas ● Melanocytes are derived from neural crest
INTERMEDIATE FILAMENT MARKERS
and will be reactive for S100 protein
● Intensity of staining for S100 is usually
Actin ● Used to identify tumors derived from inversely proportional to the melanin content
smooth, skeletal, and cardiac muscle of the tumor

Vimentin ● Melanomas and schwannomas: always Lymphomas ● Best screening marker for lymphoma is
stain positive for vimentin LCA (leukocyte common antigen), also
known as CD45

AT | DP | ST | MS | CP | PP | RC | CM 19
 Egg Albumin: not recommended as an adhesive agent
Cell ● Ki67 (MIB-1: reference monoclonal antibody
Proliferation for the demo of the Ki67) Cancer: comes from the latin word “cancrum” meaning crab
Markers ● PCNA (proliferating cell nuclear antigen) Pathology: study of disease
● Derived from two greek words:
○ “Pathos”: suffering
CONTROLS ○ “Logos”: study
Positive ● It is always advisable to use, as positive control, a Inflammation: From the Latin word “inflammare”: to set fire
Control section that is known and proven to contain the Rate of Algor Mortis: 7℉ per hour
antigen in question because absence of staining in Fixatives
a test section does not necessarily mean that the ● Biopsies:
antigen is absent in the tissue being studied ○ Heidenhain Susa: Tumor Biopsy
○ B5 Fixative: Bone Marrow biopsy
Negative ● Can be done using a parallel section from tissue ○ Bouin’s Solution: Pituitary biopsy
Control ● Omitting the primary antibody from the staining ● Smears:
schedule; or ○ Methanol: Blood and Bone marrow smear
● Replacing the specific primary antibody by an
immunoglobulin that is directed against an
○ Ethanol: Cytological smears
unrelated antigen ● Rabies diagnosis:
○ Carnoy’s Fluid
Internal ● Also termed “built-in” control ○ Acetone
Tissue ● Eliminates the variable of tissue fixation between Carnoy’s Fluid Acetone
Control specimens and controls but it contains the target
antigen
Uses: Uses:
● Preservation of ● Preservation of water-
SUMMARY chromosomes diffusible enzymes (such as
● Preservation of lymph lipases & phosphatases)
MOST RAPID glands and urgent biopsies ● Preservation of brain tissue
● Preservation of brain tissue rabies diagnosis
● Carnoy’s Fluid: most rapid fixative of all for rabies diagnosis
● Phloroglucin-Nitric Acid: most rapid decalcifying agent ● Preservation of glycogen
● Xylene/ Xylol: most rapid and most commonly used ● Post-mortem tissue fixatives:
clearing agent
○ 10% Formol-Saline
○ Formol-Corrosive (Formol-Sublimate)
MOST COMMON ● For Electron cytochemistry:
○ KPG
● Mercuric Chloride: most common metallic fixatives; ○ Acrolein
Routine fixative of choice for tissue photography ● For Electron Microscopy:
● B5 Fixatives: commonly used for Bone Marrow biopsies
○ Glutaraldehyde
● Nitric Acid: most commonly used decalcifying agents
● Alcohol: most commonly used in ascending grades or
○ Platinic chloride (PtCl3)
dehydrating agents ○ Platinic Chloride-formalin (Zamboni’s
● Xylene/ Xylol: most rapid and most commonly used fixative)
clearing agent ○ Gold chloride (AuCl)
● Paraffin Wax: most commonly used impregnating medium ○ Osmium tetroxide
● Rotary/Minot Microtome: most common types of ○ 10% BNF
microtomes that used nowadays especially for
paraffin-embedded tissues
Temperatures:
● Eosin Y: most commonly used eosin
● Mucoproteins: most common PAS-positive substances
● Flotation Water Bath: 45-50℃ (approximately 6-10
lower than the MP of wax)
BEST ● Microwave Fixation: 45-55℃ (optimum temperature)
● Paraffin Oven: 55-60℃ (must be maintained at a
● 10% BNF/NBF: Best general tissue fixative temperature 2-5°C above the MP of the paraffin
○ Best fixative for tissue containing iron pigments wax)
and for elastic fibers ● Melted Paraffin (Embedding): 5-10℃ above its MP
● 5% formic acid: Best general decalcifying agent ● Fixation:
● Ethanol: Best dehydrating agent ○ Surgical specimen (usual fixation temperature):
● Paraffin wax: best infiltrating or embedding medium Room Temperature
● Belgium Yellow: Best honing result ○ Electron Microscopy and Histochem: 0.4℃
● Neutral Red: probably the Best vital dye
● Decalcification:
○ Optimum temperature: Room Temperature
☆ BOARD EXAM RECALLS ☆ (18-30°C)
Characteristics of Glacial Acetic Acid ○ At 55°C = tissue will undergo complete
1. It solidifies at 17°C digestion within 24-48 hours
2. Causes the tissue to swell Ratio:
3. Preserves the nucleoproteins ● 20:1 (correct fixative to tissue ratio)
4. Destroys the mitochondria and Golgi bodies ● 20:1 (decalcifying fluid to tissue volume)
of the cells Rate of Penetration: Formalin = 1 mm/hr
5. Can act as a simple fixative Acid Formaldehyde Hematin may be removed by:

AT | DP | ST | MS | CP | PP | RC | CM 20
 ● Saturated alcoholic picric acid
● Alcoholic KOH

Dezenkenization (uses alcoholic iodine): used to removed

since mercuric chloride can produce black granular deposits
Von Ebner’s Fluid: recommended for teeth and small pieces
of bones

Microtomes:
1. Rocking Microtome or Cambridge Rocking
Microtome: simplest; Inventor: Paldwell Trefall
(1881)
2. Rotary Microtome or Minot Microtome: most
common type; Inventor: Minot (1885-1886)
3. Sliding Microtome: most dangerous type;
Inventor: Adams (1789)
○ Standard Sliding Microtome: most dangerous
type of sliding microtome due to movable
exposed knife
﹘ Block: Stationary
﹘ Knife: Moving
○ Base-Sledge Microtome:
﹘ Block: Moving
﹘ Knife: Stationary
4. Rotary Rocking Microtome:
5. Vibrotome: for enzyme demonstrations
6. Ultrathin Microtome: for EM; uses Diamond Knives
or broken plate glass
7. Freezing Microtome: Rapid Diagnosis; Inventor:
Queckett (1848)

Honing & Stropping:

● Honing: Heel to Toe
● STropping: Toe to Heel

Clearance & Bevel Angle:

● Clearance Angle: 0-15° or 5-10°
● Bevel Angle: 27-32°

Congo red: gold standard for amyloid demonstration

Mercuric Oxide Sodium Iodate

ripening agent of Harris ripening agent of Ehrlich’s

Hematoxylin Hematoxylin

Microtome Knives

Microtome Knives Usual Length Description

Plane concave 25mm One side: flat

Other side: concave

Biconcave 120mm Both sides:

concave

Plane wedge 100mm Both sides: straight

☆ SAMPLE BOARD EXAM QUESTIONS☆

“All of the following contain glacial acetic acid except:
Cytoplasmic fixatives”

AT | DP | ST | MS | CP | PP | RC | CM 21