Enclonar, Kimberly / MLS 3A

▪ 10% neutral buffered formalin

Fixation, Decalcification & ▪ Heidenhain's Susa
Dehydration ▪ Formal sublimate (formal corrosive)
October 21, 2020 ▪ Zenker's solution
Gabriel Ponce de Leon, RMT, AMT, ASCPi, MS ▪ Zenker-formal (Kelly's solution)
▪ Bouin's solution
Histology Procedure ▪ Brasil's solution
1. Specimen Accessioning o Cytologic Fixatives
o Viability of sample ▪ Nuclear
o Pre-fixation □ Flemming's fluid
2. Gross Examination □ Carnoy's fluid
o Orient tissue in its original anatomical structure □ Bouin's fluid
o TAHBSO (total abdominal hysteroctomy - □ Newcomer's fluid
bilateral salpingoophorectomy) □ Heidenhain's Susa
o Organ or organ system ▪ Cytoplasmic
o Tubular or solid □ Flemming's fluid without acetic acid
3. Tissue Fixation □ Kelly's fluid
o Start of tissue biopsy □ Formalin w/o post-chroming
o A portion of the tissue only □ Orth's fluid
o Formaldehyde □ Regaud's fluid (Muller's fluid)
4. Tissue Processing ▪ Histochemical
o Dehydration □ 10% formal saline
o Clearing □ Absolute ethyl alcohol
o Impregnation □ Acetone
5. Tissue embedding □ Newcomer's fluid
6. Tissue sectioning
o Before: deparaffinization A Aldehyde Fixative
o After: fishing out in a float out bath • Formaldehyde
7. Slide staining o 37-40% stock solution
▪ Liquid form
Fixation ▪ Distilled water as diluent
o 10% working solution
• First and most critical step
o If tissue is too brittle, there will be a problem in o Advantage: cheap, easy to prepare and readily
cutting available
o At least 3 representative samples o Disadvantage: May produce considerable
shrinkage of tissues
• Ideal time to perform fixation: 20-30 minutes after
▪ May cause brittle tissues
interruption of blood supply
o Prevents autolysis • 10% Formol Saline
o For CNS tissues
• Correct fixative-tissue ratio - 20:1
o Advantage: Preserves enzymes and
• Usual fixation temperature: room temp
nucleoproteins, demonstrates fats and mucin
• Primary aim:
o Disadvantage: slow fixative
o To preserve the morphological and chemical
integrity of the cell in as life-like manner as • 10% buffered Neutral Formalin
o For post-mortem and research specimen
possible
o Advantage: Best fixative for tissue containing
• Secondary aim:
o To harden and protect the tissue from the iron pigments
o Disadvantage: It is longer to prepare.
trauma of further handling
• Formal Corrosive (FOR-SMOL-CHLOROSIVE)
o For routine post-mortem tissue
Classification of Fixatives
o Adv: Penetrates small tissues
• Physical fixative
o Heat o Disadv: Forms mercuric chloride deposits
o Freezing • Glutaraldehyde (GluEM)
o For electron microscopy
• Chemical Fixatives
o Simple fixatives o Adv: It preserves cellular structures
o Disadv: It is more expensive
▪ Aldehydes
□ Formaldehyde
□ Glutaraldehyde B Metallic Fixative
▪ Metallic fixatives • Chloride
□ Mercuric chloride o Most common metallic fixative
□ Chromate fixatives o May produce black granular deposits on tissues
▪ Picric acid (except SuSa)
▪ Acetone • Chromate
▪ Acetic acid o Preserves chromatin tissue
▪ Alcohol • Lead fixative
▪ Osmium tetraoxide o For acid mucopolysaccharide
▪ Osmic acid • Mercuric chloride
o Compound fixatives o Zenker's Fluid (w/ glacial acetic acid)
o Microanatomical fixatives (ZenGACTRICH)
▪ 10% formal saline ▪ Adv: Recommended for trichrome staining
 Enclonar, Kimberly / MLS 3A
▪ Disadv: poor penetration • Inhibits hematoxylin and makes counterstaining
o Zenker-Formol (Kelly's solution) difficult
▪ Adv: Excellent for pituitary gland, bone • Produces black precipitate (osmic oxide)
marrow, spleen and liver • Prevention: add saturated aqueous HgCl2
▪ Disadv: Brown pigments are produced • Remedy: dissolve in cold water
▪ Brown pigments can be dissolved in • Flemming's
saturated picric acid or NaOH. o For nuclear structure
o Heidenhain's Susa • Flemming's with acetic acid
▪ For tumor biopsies of the skin o For cytoplasmic structures
▪ Adv: penetrates and fixes tissues rapidly
and evenly G Trichloroacetic acid
▪ Disadv: RBC preservation is poor • Both a fixative and decalcifying agent
o B5 Fixative (B5BMB)
▪ For bone marrow biopsies H Acetone (Asotone)
▪ Adv: rapid fixation can be achieved in 1 1/2 • Fixes brain tissues for the diagnosis of rabies
- 2 hrs
▪ Disadv: Over fixation hardens tissue I Heat Fixation
• Chromate • Direct flaming
o 1-2% Chromic acid o For bacterial smear
o 3% potassium dichromate (POTA DIMITO)
• Microwave fixation
▪ preserves mitochondria o Optimum temperature: 45-55C
o Regaud's (Mauller's)
▪ mitochondria, golgi apparatus
o Orth's fluid Decalcification
▪ For Rickettsia • Removal of calcium
▪ For study of early degenerative diseases • Bone, teeth, etc
• Lead Fixative • Recommended ratio of 20:1
o Alcian Blue (Alcid) • At 37C, impaired nuclear staining of Van Gieson's
▪ For acid mucopolysaccharide stain for collagen fibers
• At 55C, tissue will undergo complete digestion within
C Picrate Fixative 24-48 hrs
• Picrates are formed upon protein; precipitates are • Optimum temperature: RT (18-30C)
soluble in water; • Ideal time: 24-48 hrs
• Hence tissues must be first rendered insoluble by
direct immersion in 70% ETOH Methods of Decalcification
• Picrates fixative must never be washed in water Acid decalcifying agents
before dehydration • Nitric Acid
• 70% ethyl alcohol - 5% sodium thiosulfate - running o Most common
water o Fastest decalcifying agent
• Bouin's solution o Used for large or heavily mineralized cortical
o For embryos and pituitary biopsies bone specimen (aqueous nitric acid solution
o Adv: excellent for preserving soft and delicate 10%)
tissues o Examples:
o Disadv: penetrates larges tissues poorly ▪ Formol nitric
• Brasil's solution (BRAniGLYCO) □ urgent biopsies
o For excellent fixative for glycogen ▪ Perenyi's
o Adv: less messy □ tissue softener and decalcifying agent
▪ Phloroglucin-nitric acid
D Glacial Acetic Acid (GAA-Nucleo) □ Most rapid decalcifying agent
• Adv: Fixes and precipitates nucleoproteins • Hydrochloric acid
o Von Ebner's solution
• Disadv: destroys mitochondria and golgi apparatus
▪ Teeth and small pieces of bone
E Alcoholic Fixative • Formic acid
o Fixative and decalcifying agent
• Methyl alcohol o Formic acid-sodium citrate solution
o For dry and wet smears
▪ Recommended for autopsy materials, bone
• Isopropyl alcohol
marrow, cartilage and tissue studied for
o For fixing touch preparations
research purposes
• Ethyl alcohol
o 70-100% • Trichloroacetic acid
o Does not require washing out
• Carnoy's (fastest Car) o Is suitable for small spicules of bone
o most rapid fixative, fixation time 1-3hrs
o Weak decalcifying agent
• Newcomer's fluid
o Fixes mucopolysaccharide • Sulfurous
o Very weak decalcifying agent
• Chromic acid (Flemming's fluid)
F Osmium Tetraoxide o Fixative and decalcifying agent
• Should be kept in a dark-colored chemically clean
• Citric acid-citrate buffer solution
bottle to prevent evaporation and reduction by o Permits excellent nuclear and cytoplasmic
sunlight or organic matter staining; action is too slow for routine purposes
 Enclonar, Kimberly / MLS 3A
Ion exchange resins with acid and decalcifying fluids o Utilized for urgent biopsies
• The removal of calcium ions from the decalcifying • Dioxane (diethylene dioxide)
fluid by the resins leads to quicker and more efficient o Dehydrating agent and a clearing agent
decalcification o Makes use of Graupner's method and
Weiseberger's method as a time schedule for
Electrolytic decalcification dehyration
• The speedier decalcification without damage to the • Cellosolve (Ethylene glycol monoethyl ether)
cytological features and staining o Combustible at 110-120F and are toxic by
• Drawback: heat produced in the process may cause inhalation, skin contact and ingestion
the charring of specimen in the process • Triethyl phosphate
• Tetrahydrofuran (THF)
Chelating agents o Dehydrating agent and a clearing agent, produce
a good staining results
Measuring the Extent of Decalcification • Carbon tetrachloride
• Physical or Mechanical Test o When heated produces a poisonous gas known
o Done by touching or bending the tissue with the as "Phosgene"
fingers to determine the consistency of tissues. • Methyl benzoate and methyl salicylate
o Alternative method is done by pricking the tissue o Slow acting clearing agents that can be used
with fine needle or probe when double embedding techniques are
• X-ray or Radiological Method required
o Most ideal, most sensitive and most reliable yet
it is the most expensive
o Has the ability to detect even the smallest focus
of calcium which appears opaque in an X-ray
plate
• Chemical Method (Calcium Oxalate Method)
o Simple, reliable and convenient method
o Involves the detection of calcium in acid
solutions by precipitation of insoluble calcium
hydroxide or calcium oxalate

Post-Decalcification
Can be done by:
• Neutralization
o Lithium carbonate solution or 5-10% aqueous
sodium bicarbonate
• Rinsing - tap water
• Blotting - tap water before transferring it into
dehydrating fluids
• Dehydration - 70% alcohol

Tissue Softeners
• Perenyi's fluid
o Decalcifying agent and tissue softener
• 4% aqueous phenol solution
o Immersion of tissue samples for 3 days
• Molliflex
o Commercially prepared
• 2% HCl or 1% HCl in 70% alcohol

Dehydration
• Aim: remove fixative and water from tissues and
replaced them with dehydrating agents
• In most instances, dehydration starts by placing the
specimen from 70% ethanol in water, progressing
through 95% ethanol to 100% ethanol

Commonly used:
• Alcohol (most common)
o Ethyl alcohol
▪ For routine dehydration
▪ Best dehydrating agent because it is fast-
acting
o Methyl alcohol
▪ For blood and tissue films
o Butyl alcohol
▪ For plant and animal microtechniques
• Acetone
o rapid acting dehydrating agent