HISTOPATHOLOGIC

TECHNIQUES AND
MEDTECH LAWS
Histology
• 3 GERM CELL LAYERS

GERM CELL LAYER TISSUE

Ectoderm Nervous tissue

Epithelial tissue

Endoderm Epithelial tissue

Mesoderm Connective tissue

Muscular tissue
Epithelial tissue
Epithelial tissues
A. Covering Epithelia
According to cellular arrangement:
• Simple
• Stratified
• Pseudostratified

According to cellular shape:

• Squamous
• Cuboidal
• Columnar
• Transitional
Epithelial tissues
Epithalial tissue Example
Simple squamous Bowman’s capsule
Endothelium
Loop of Henle
Lung alveoli
Simple cuboidal Ducts of glands
Walls of thyroid follicles
Simple columnar Gallbladder (non-ciliated)
Uterine tube (ciliated)
Stratified squamous Skin epidermis (keratinized)
Vagina
Esophagus
cervix
Stratified cuboidal Ducts of sweat glands
Stratified columnar Male urethra
Transitional Urinary tract
Pseudostratified columnar Female reproductive tract (non-ciliated)
Trachea (ciliated)
Note:
• CHRONIC SMOKERS
METAPLASIA: reversible
Change of tracheal tissue (ciliated pseudostratified
columnar to stratified squamous)
Connective tissues
General
• Loose connective tissue (wharton’s jelly, bone marrow, embryo)
• Dense connective tissue (capsule of organs, tendons, stroma of
cornea)

Special
• Cartilage: hyaline (intervertebral disc); elastic (external ear,
epiglottis)
• Bone: cancellous (epiphysis of long bones); compact (diaphysis of
shaft)
• Blood
• Lymph
Connective tissues
Pathologic changes
 Fibrin
 Fibrinoid
 Hyalin: PAS
 Amyloid: Gram’s iodine stain, Congo red

 All are EOSINOPHILIC in H and E stain

Connective tissues
Collagen – major component of CT
 Collagen stains:
a) Van Gieson’s stain
b) Masson’s Trichrome
c) Mallory’s aniline blue
d) Azocarmine
e) Kraijan’s aniline blue
Inflammation

1. Rubor- “redness”

2. Tumor -“swelling”

3. Calor- “heat”

4. Dolor- “pain”

5. Functio laesa- “diminished function”

Inflammation
A. According to duration:
Acute
Subacute
Chronic

B. According to exudate:
Serous: pulmonary TB
Fibrinous: diphteria, rheumatic pericarditis, pneumonia
Catarrhal: hypersecretion of mucosa
Hemorrhagic: bacterial infections
Suppurative: pus
Changes in cellular growth patterns
 Retrogressive: smaller than normal
Developmental defects
• Aplasia: incomplete development; mass of fatty/fibrous tissue
• Agenesia: non appearance of an organ
• Hypoplasia: failure of an organ to reach its full mature size
• Atresia: failure of an organ to form an opening
Atrophy
Decrease in size of normally mature tissue/organ
Reduction in cell size
Decrease in total number of cells
• Physiologic
• Pathologic
Changes in cellular growth patterns
 Progressive: larger than normal
Hypertrophy: increase in cells size
Hyperplasia: increase in the cell number
• Physiologic
• Pathologic

 Degenerative: abnormalities
Metaplasia: one adult cell to another; reversible (eg. Chronic smokers)
Dysplasia (atypical hyperplasia): changes in structural component;
reversible
Anaplasia (differentiation): adult cell to more primitive cell (eg. Cancer)
Neoplasia (tumor): proliferation without control (eg. cancer)
Death
Cellular death
Apoptosis
Necrosis
• Coagulation: “tombstone formation”
• Liquefaction: pus
• Caseous : yellow cheesy materials
• Gangrenous: sulfide gas
• Fat: white chalky ppt.
Death
Somatic death
Primary: circulatory, respiratory and CNS failure
Secondary:
1. Algor mortis
FIRST
Cooling of the body
2. Rigor mortis
Stiffening
3. Livor mortis/Postmortem lividity
Purplish discoloration
4. Postmortem clot
5. Dessication
6. Putrefaction
7. Autolysis
Death

Postmortem clot Antemortem clot

Settling of RBC Granular and friable

Chicken fat Not readily detachable from the blood

Vessel
Currant jelly
Seldom assumes the shape of Vessel
Assume the shape of the vessel
Exfoliative cytology
Non-gynecologic specimen:
• Sputum
3 consecutive morning sputum
Alveolar macrophage
• Bronchoalveolar lavage/wash
Pull apart technique
• Peritoneal, pleural and pericardial fluids
300 units heparin/ 100 mL aspirate
• Gastrointestinal lavage
• Gastric cancer
• Urine
Urothelial malignancy
Atleast 50 mL
Second urine is preferred
Exfoliative cytology
Gynecological specimen
• Pap smear/ papanicolaou smear
Upper lateral third of the vaginal wall
 Ectocervix: stratified squamous (ayer’s spatula)
 T-zone- transformation
 Endocervix: simple columnar (endocervix)

Cells found in cervico-vaginal smears:

 Mature superficial cell: increased by estrogen; “true acidophilia”; pyknotic
nucleus
 Intermediate cell: increased during pregnancy; basophilic cytoplasm with
vacuoles
o Navicular cells: boat shaped, with folds and curls
o Pregnancy cells:double walled; nucleus is pushed aside
 Parabasal cell: late menopausal; “sunny side up egg”
 FERNING
 “palm leaf pattern”
Exfoliative cytology  Salt crystal
formation
• Endometrial cell  Ovulation and
• Endocervical glandular cell fertility
 “honeycomb appeacrance”
• Basal cells
 Found before puberty and after menopause
• Doderlein bacillus
 Lactobacillus acidophilus
• Candida albicans
• Trichomonas vaginalis
 “strawberry cervix”
• Gardnerella vaginallis
• Koilocytes
 “wrinkles prune”
 HPV
Exfoliative cytology
Fixation
A. Fluid specimens
 50% alcohol(pleural, peritoneal); 7-% alcohol(sputum); 95%
alcohol(urine, bronchial, gastric)
Saccomano’s fixative (50%ethanol and 2%carbowax)
Centrifugation: 2000 rpm for 2 mins

B. Smears
Equal parts of 95% ethanol and ether: BEST
95% ethanol: COMMONLY USED
Spray fixative: 1 foot away
HISTOPATHOLOGIC TECHNIQUE
 FRESH TISSUE EXAMINATION
a) Teasing/dissociation- isotonic solution in a watch glass
b) Crushing/ squash preparation- vital stain
c) Smear preparation
d) Frozen section
 Rapid diagnosis (Lipids, Nervous tissue)
 Fixative: formol calcium
 Freezing: liquid N (most rapid)
 4 deg.C for 18 hours
Preparing frozen sections
1. Cold knife procedure
Knife: -40 to -60 deg.C
Tissue: 5 to -10 deg.C
Environment: 0 to -10 deg.C

2. Cryostat procedure/ cold microtome

OPTIMUM WORKING TEMP: -18 to -20 deg.C
Microtome of choice: rotary microtome
Preserved tissue examination
1. Fixation
2. Dehydration
3. Clearing/ dealcoholization
4. Impregnation/ infiltration
5. Embedding
6. Trimming
7. Sectioning/cutting
8. Staining
9. Mounting
10. Labeling
I. Fixation
• FIRST AND MOST CRITICAL
• Preservation
• Optimum pH 6-8
• 20:1 fixative to tissue ratio
• Room temperature
• Types of fixatives:
Composition
 Simple fixative
 Compound fixative
Action
 Microanatomical (10% formol saline, Heidenhein’s susa, Bouin’s, Zenker’s slution,
Zenker-formol , formol sublimate)
 Cytological
1. Nuclear fixative (with glacial acetic acid)
Flemming’s, Carnoy’s (chromosomes), Newcomer’s

2. Cytoplasmic fixative (without glacial acetic acid)

Flemming’s without Gac, Helly’s, Formalin with post
chroming, Regaud’s (Muller’s), Orth’s

3. Histochemical fixative
Absolute ethanol, Acetone, Newcomer’s
Aldehyde fixatives
• Formaldehyde
37 to 40% stock solution
10% working solution; unstable
1 mm/hr
• 10% formol saline
CNS tissues
• 10% BNF (buffered neutral formalin)
BEST GENERAL FIXATIVE
Best for tissue containing iron pigments
• Formol- sublimate/ formol- corrosive
Contain mercuric chloride
• Glutaraldehyde
Plasma protein
• Formol calcium
lipids
Metallic fixatives
• Mercuric chloride
MOST COMMON (metallic fixative)
May produce black granular deposits except Heidenhein Susa
Zenker’s, Zenker-formol (helly’s), Heidenhein Susa(biopsy of skin),
Schaudinn’s fluid(stool), B-5 fixative(bone marrow)

• Chromate
Chromic acid, potassium dichromate, Regaud’s(Muller’s)
Orth’s- for riskettsia; study of early degenerative processes

• Lead
ACID MUCOPOLYSACCHARIDES (wharton’s jelly, umbilical cord)
Picrate fixatives
• Highly explosive
• Yellow staining of tissues
• MUST NEVER be washed with water before dehydration
• Bouin’s solution- for fixation of embryos
• Brasil’s solution

Glacial Acetic Acid

 Fixes nucleoproteins
Alcoholic fixatives
• Methanol- blood smears and bone marrow tissues
• Ethanol, Isopropyl alcohol
• Carnoy’s fluid- MOST RAPID (1 to 3 hours)
• Alcoholic formalin (Gendre’s fixative)- sputum

Osmium tetroxide fixatives

• Should be kept in amber bottle
• Inhibits hematoxylin
• Produce black ppt (osmic oxide)
• May cause blindness
• Flemming’s solution
Trichloroacetic acid
Acetone
 dx. of rabies
Heat fixation
 bacteriology
 Fixative pigments Remedy Appearance

Acid formaldehyde hematin • Picric acid Black/ brown granules

(formic acid + Hgb) • 1% KOH

Mercuric Chloride Alcoholic iodide Black granular deposits

Chromate Acid alcohol Fine yellow deposit

Osmium tetroxide Cold water Black ppt

Decalcification
• 20:1 ratio
• Optimum temp: 18-30 deg.C
• 24-48 hours
• Dense bone: 14 days
• Decalcifying agents:
a) Acids
 Nitric acid: MOST COMMON (Perenyi’s fluid-tissue softener; Phloroglucin-Nitric acid-
MOST RAPID)
 Formic acid: both fixative and decalcifying agent (5% formic acid-BEST GENERAL
DECALCIFYING AGENT)
 Hydrochloric acid: (Von Ebner’s fluid)
b) Chelating agents
c) Ion exchange resin
d) Electron ionization
II. Dehydration
10:1 ratio
Remove fixative and water
Increase strength of tissue
 Alcohol- MOST COMMON; progressively increasing
concentration
oEthanol: BEST DEHYDRATING AGENT
oMethyl alcohol: blood and tissue films
oButyl alcohol: plants and animal microtechniques
 Acetone: both fixative and dehydrating agent
 Dioxane (Diethylene dioxide): both dehydrating and clearing
agent
 Tetrahydrofuran: both dehydrating and clearing agent
• Additives:
4% phenol to each 95% ethanol bath
Anhydrous copper sulfate
Both dehydrating and indicator for incomplete dehydration
Water is present: white turns blue
III. Clearing
• DEALCOHOLIZATION
• Miscible to both dehydrating and impregnating fluid

 Xylene/Xylol
 Toluene
 Chloroform: Toxic; used for tough tissues
 Aniline oil: delicate tissues
IV. Impregnation
• 25:1 ratio
• Aka. Infiltration
• Same medium for embedding

 Paraffi n
 MOST COMMON AND THE BEST INFILTRATING/EMBEDDING MEDIUM
 Not for fatty tissues
 Temp of the paraffin oven: 55-60 deg.C (2-5 deg.C above melting pt. of paraffin wax)
a) Paraplast: 56-57 deg.C, more elastic then paraffin; for bones and brain
b) Embeddol: 56-58 deg.C; less brittle and less compressible than paraplast
c) Bioloid: recommended for eye specimen
d) Tissue mat: contains rubber
e) Ester wax: 46-48 deg.C, harder than paraffin, not water soluble, can be used without prior clearing
f) Carbowax: water soluble, does not require dehydration and clearing, for enzyme histochem,
 Celloidin
Purified form of nitrocellulose
For specimens with large hollow cavities, hard and dense tissues (bones
and teeth); embryo
• Wet celloidin: bones and teeth
• Dry celloidin: whole eye
 Gelatin
For enzyme studies
For frozen sections
Does not require dehydration and clearing

 Plastic
Epoxy, polyester, acrylic
V. Embedding
• blocking
• Orientation
• Temperature of melted paraffi n: 5-10 deg.C above melting pt.
• Cooled rapidly in a freezer -5 deg.C
• Atleast 2 mm of wax should surround the tissue
VI. Trimming
• Process of removing excess wax after embedding
• Blade or knife is used

Microtome knife:
1. Plane- concave: 25mm, celloidin embedded tissue on a sliding
microtome
2. Biconcave: 120mm, paraffin embedded tissue on rotary microtome
3. Plane-wedge: 100mm, frozen sections; tough paraffin embedded tissue
on base-sledge microtome or sliding microtome
Bevel angle: 27 to 32 degree
Optimum cutting angle is 14 degree
Sharpening of knives
• Honing
Removal of gross nicks and blemishes
Hone :belgium yellow, arkansas, fine carborundum
EDGE FIRST, HEEL TO TOE

• Stroping
Removal of burr and iregularities
Strope: horse leather
Final polishing of the knife
EDGE LAST, TOE TO HEEL
VII. Sectioning
• Cutting or microtomy
• Routine: 4-6 u
• Frozen section: 10-15 u
• Enzyme microscopy: 0.5 u

Floatation bath
45-50 deg.C
6 to 10 deg.C below melting pt of paraffin wax

Adhesive agent:
• Egg albumin: crystal of thymol is added
• 3-aminopropyltriethoxysilane: BEST ADHESIVE AGENT
Microtome:
a) Rocking microtome/ Cambridge
Simplest, Paldwell Trefall in 1881
b) Rotary microtome/ Minot
Minot in 1885-1886
c) Sliding microtome: MOST DANGEROUS
Adams in 1789
Base sledge microtome: block holder-moving, knife-stationary
Standrard sliding microtome: block holder-stationary, knife-moving
d) Rotarty rocking microtome
e) Vibrotome: enzyme demonstration
f) Ultrathin microtome: enzyme microscopy, diamond knives, fixed in
osmium tetroxide, embedded in plastic
g) Freezing microtome: Queckett in 1848
VIII. Staining
• Chromophores- “color bearers”
• Auxochrome- “increasers”
• Dye modifier- susbtance that affect either the color or properties of
the dye
• Lake- “stain-mordant-tissue complex”
• Orthochromatic staining
• Metachromatic staining
• Progressive staining
• Regressive staining
• Chromogen
• Dye
H and E staining
• Hematoxylin
Natural dye
Primary stain
Heartwood of mexican tree “Hematoxylin campechianum”
Hematein- active coloring substance
A. Alum hematoxylin
• Routine
• Mordant: potash alum
• Red nuclear stain
B. Iron hematoxylin
• Mordant: iron salts
• Weigert’s- ferric chloride; Heidenhains- ferric ammonium sulfate
C. Tungsten hematoxylin
• Mallory’s PTAH
D. Copper hematoxylin- spermatogenesis
• Eosin
Red acid dye
Counter stain
a) Eosin Y-MOST COMMONLY USED
b) Eosin B
c) Ethyl Eosin
H and E staining steps (regressive)
1. Xylol (2 changes)- deparaffi nization
2. Descending grade of alcohol- rehydration
3. Water
4. Harris hematoxylin
5. Water
6. Acid alcohol- differentiator
7. Ammonia water (scott’s tap water)- blueing
8. Water
9. Eosin Y
10. Ascending grade of alcohol
11. Xylol- clearing
12. Mount then label
PAP smear staining
1. Fix with 95% Ethanol
2. Harris hematoxylin
3. Acid alcohol
4. Blueing
5. OG-6- stains cytoplasm of mature superficial cells
6. 70-95% ethanol for washing
7. EA-36/50/65- stains cytoplasm of immature cells (intermediate,
parabasal)
8. Dehydrate
9. Xylol
10. Mount and label
IX. Mounting
• Refractive index of glass: 1.524
Resinous media (greater or equal to 1.518)
 DPX (1.532)
 XAM (1.52)
 Canada balsam/ abus balsamea (1.524)
 Clarite (1.544)
Aqueous media ( for lipids)
 Glycerin
 Farrant’s medium/ Gum Arabis (1.43)
 Apathy’s medium (1.52)
 Brun’s fluid- for frozen sections
MEDTECH LAWS

REPUBLIC ACT 5527

PHILIPPINE MEDICAL TECHNOLOGY ACT
of 1969
JUNE 21,1969
32 sections
Council of Medical Technology Education
Chairman: Director of higher education
Vice- Chairman: Chairman of PRC
Members:
 Director of Bureau of Research and Laboratories of the DOH
 Chairman and two members of the Board of Medical
Technology
 Representative of the dean of schools of MT and PH
 President of Philippine Society of Pathologist
 President of PAMET

NOTE: PAMET PRESIDENT 2014

Romeo Joseph J. Ignacio, RMT
Medical Technology Board
Chairman: Pathologist
Two members: appointed by the President of the Republic of
the Philippines

 3 years in position
Minimum required Course
• 4 years
• 12 months satisfactory internship in accredited lab

CHED memorandum order #14 of 2006

Title: Policies, Standards and guidelines for Medical
Technology Education
6 months internship
To pass the MT board examination
• Over all average of atleast 75% provided that:
No rating below 50% in any of the major subjects
Has not failed in at least 60% of the subjects
computed according to their relative weight

• Failure to qualify for the 3 rd time: 12 months refresher

course in an accredited laboratory
Republic Act 4688
• Clinical Laboratory Act of 1966
• June 18, 1966
• Classification of Clinical Laboratories

Ownership Function Institutional Service

character capability

 Government  Clinical  Institution  Primary

 private  Anatomic based  Secondary
 Free-  Tertiary
standing
General Laboratory (by service capability)
Primary Secondary Tertiary

 Routine hematology Services of primary plus: Service of Primary and

 Qualitative platelet secondary plus:
determination  Routine clinical
 Routine fecalysis chemistry  Special chemistry
 Routine urinalysis  Quantitative Platelet  Coagulation procedures
 Blood typing (for determination  Immunology
hospital based)  Cross matching  Microbiology- culture
 Gram staining and sensitivity
 KOH
Republic Act 7719
• National Blood Services Act of 1994
• May 5, 1994
• BLOOD SERVICE FACILITY

Ownership Institutional Service Capability

Character
 Government  Hospital based  Blood station
 Private  Non-hospital  Blood collection
(hospital based based unit
only) (government  Blood bank
owned or PNRC  Blood center
owned only)
Blood Service Facility (by service capability)
BLOOD • Advocacy and promotion of voluntary blood donation
STATION (BS) • Provision of WB and PRBC
• Storage, issuance, transport and distribution of WB and PRBC
• Compatibility testing of red cell units if hospital based

BLOOD • Advocacy and promotion of voluntary blood donation

COLLECTION • Recruitment, selection and screening of voluntary blood donors
UNIT (BCU) • Compatibility testing of red cell units if hospital based

BLOOD BANK • Advocacy and promotion of voluntary blood donation

(BB) • Recruitment, selection and screening of voluntary blood donors
• Storage, issuance, transport and distribution of blood
components
• Compatibility testing, Coomb’s test, Antibody screening
• Investigation of Transfusion reaction

BLOOD • Blood bank capabilities

CENTER (BC) • Testing of TTIs
• Processing and provision of blood components
Republic Act 8504
• Philippine AIDS Prevention and Control Act
• February 13, 1998
• Philippine National AIDS Council
Composed of 26 members

AIDSWATCH
Monitors magnitude of HIV infection in the Philippines
Republic Act 9288
• Newborn Screening Act of 2004
• April 7, 2004
• Newborn screening shall be performed after 24 hours
of life but not later than 3 days from complete
delivery of the newborn
• Newborn in the ICU may be exempted from the 3 day
requirement but must be tested by 7 days of age.
Disorders screened
Disorder Effects

Congenital hypothyroidism Severe mental retardation

Congenital adrenal Death

hyperplasia

Galactosemia Death and Cataracts

Phenylketonuria Severe mental retardation

G6PD deficiency Severe anemia, kernicterus

Others:
• R.A 9165
Comprehensive Dangerous Drugs Act of 2002
June 7, 2002
• R.A 9482
Anti-Rabies Act of 2007
May 25, 2007