Histopathology and Laboratory Management

PRE AND POST ANALYTICAL

PROCEDURES RECEIVING THE SPECIMEN
Subtopic: Receiving, Accessioning, & Releasing of - it is possible to submit another test aside from
Results biopsy meron din for cytology or cell block.
- Like halos tanan test sa histopath maperform or
PRE-ANALYTICAL ipaperform ng surgeon
- Always check the label (completely labeled)
- Cross-check if the information in the request - One patient can have more than 1 specimens
matches/correlates the labels in the specimens submitted in the lab
- Double check if the surgeon or the attending
physician declared the submitted specimen 1. CHECK THE DOCUMENTS
needed for sampling. - Check the documents received and correlate it
- Recommended histopath containers: must be with the specimens received.
locked by twisting. - Manner of submission can be:
o Request is folded and is inside the
ziplock along with the specimen
o Hospital property requests: in-patient
o Other hospitals requests or prescriptions
(Rx): outpatient
- Histopathology Request:
o Name
o Date Requested
o Date of Birth
o Admission/Case No. (in-patient)
o Address of Patient*
o Age/Sex
o Contains 10% neutral buffered formalin. o Civil Status
o Specimens must be directly proportional o Room No.
to the specimens. o Referring Department*
 Ex. Skin Punch Biopsy o Clinical Impression – initial diagnosis
(performed by derma patho) (important: the pathologist will correlate
 Patients have allergies and the the findings to the impression)
doctored suspected that it is o Operation/Procedure Performed
malignant hence they will collect o Specimen – must be indicated.
the sample. o Brief Clinical Data – aid the pathologist
 Buslotan ang affected area then to arrive to his diagnosis since he can
submit to histopath for biopsy to correlate it to the patients signs and
examine the problem in a tissue symtptoms
level. o Tentative/Pre-Operative and/or Post-
 Skin punch biopsy is around 0.2
Operative Findings
cm and should be contained in
o Additional Request/s or Special Stain to
a small container that is
be done.
proportional to its size.
o Requesting Physician name and
o USUAL SET-UP IN THE PH
signature
 Some labs cannot afford the
o * - (depends on hospital)
containers shown above hence
they use zip locks or plastics - Specimen Container:
and can be accepted as long as o Full patient name
it is properly labeled and the o Medical Record Number
formalin does not leak (the o Age & Sex
specimen might not be fixed o Type of Specimen
properly due to leakage) o Date & Time of Collection
- Samples must be submitted with pre-fixed with o Location of the patient – so that we can
formalin. know saan galling ang specimen and

JLLD 1
Histopathology and Laboratory Management

para sa due date (5 days TAT; some  There are referral letters from
hospital has 1 month TAT) other hospitals directed to the
doctors, so the letters must be
collected so that we can know
- Specimen Rejection Criteria kung unsa na na work up ang
o Dili pasabot na dili na gyud dawaton buhaton ani na patient
ever 2. SPECIMEN CHARGING
 Ex. Appendix must be accepted - In this process, this is the time that you can
but we have to call the double check the specimens received. Please
watcher/doctor or anyone take note also the date and time and the person
related to the patient so that we who received the specimen. You may also fill
can explain or clarify ang mg out a claim stub to be given to the
ana wrong at wala napasok sa watcher/nurse/patient.
ating criteria - Different hospitals have different charging:
 Mahirap magpa-recollect so received three specimen
tanggapin na lang but if we will o 1 – uterus
be based it to the book it should o 1 – left and right fallopian tube
be rejected – still depends on o Sizes of specimen:
lab protocol.
 Fallopian tubes = small biopsy
o Unlabeled or wrong label on the
 Uterus = large biopsy
specimen o Charging of specimens: pila mabayaran
 Can be accepted, but in the
sa patient
result form you must write that
o Ma-incorporate pila ka specimen ang na
the specimen is unlabeled.
receive in case na need mag back track
o Incomplete patient information/clinical
- Transaction Slip
history
o Billing information
 There are times when
o HIS: nurses can input some remarks
pathologists will ask for
additional information because additional remark is what type of
there are cases that is hard to specimens
diagnose.  One big container + many
o Left unfixed or unrefrigerated for an specimen = extra large tissue
biopsy charging
extended period.
- Claim Stub
 Esp if biopsy fixed sample is a
o Name
must.
o Putrefied or autolyzed specimens o Age/Sex
o Damaged specimen or broken slides o Room no.
o Insufficient sample for processing o Examination
 In real life situation, no matter o Additional Test
how small the amount the o Date Requested & Due Date
specimen is, we must process o Prepared by (sino nag receive siya nag
it. prepare)
 And be careful in processing it  This can be noted in a surgical
because it can be a specimen pathology worksheet
loss.
o Spilled or contaminated specimens 3. ACCESSIONING
 Containers must be full of - Assign the laboratory number/accession
formalin to measure that the number unique to that patient.
specimen is fixed properly
o Failure of the requesting physician to HOW TO ACCESSION?
enter the request in the computer. - Choose the appropriate logbook.
o Empty containers without the specimen o Surgical Pathology (e.g. biopsy)
or form o IHC: Immunohistochemical (further
o Referral Pathology consultation material evaluation right after the surgical
without Histopathology report or pathology if the doctor request for it)
referring hospital. o Cytology/Cell Block (e.g. body fluids)

JLLD 2
Histopathology and Laboratory Management

o Pap’s Smear (Ob-gyne collects and the  2020 = 20

anatomic location is in cervix)  143: Specimen control number
o ROC: Referral of Case (2nd opinion;  143rd specimen of the
sample from the other hospital should year
be retrieved, the cell block or tissue  Must be carefully written
block is retrieved) (Retrieval of biopsy and di pwede mamali bc
lasts for 10 years) it will be used all
o FNAB: Fine Needle Aspirate Biopsy throughout the process
(usually in Thyroid)  A: If more than one sample in
o FSB: Frozen Section Biopsy (skills is a one patient
must since it has a different microtome,  A1/ A2: number of
temperature monitored, sample is not samples taken from one
embedded in wax, but we use freezing organ.
medium, so it is hard to cut) 4. LABEL THE DOCUMENTS AND THE
o Rectal Biopsy (e.g., Schistosomiasis; SPECIMEN WITH ITS CORRESPONDING
prepared through crushing/squashing) ACCESION NUMBER
 Squashing/Crushing (0.1 cm) - Double check the patients name and
 Teasing or dissociation accession number
of sample
 Collect small portion of
the teased tissue.
 Add methylene blue
stain or NSS
 Use a cover slip or
another slide to gently
crush or squash the
small tissue.
o PBS: Peripheral Blood Smear (to check 5. PREPARE A WORKSHEET
- Name
abnormalities in blood before
- Accession number
proceeding to BMA and is read by
- Age/Sex
hematologists or pathologists)
- Due Date
o BMA: Bone Marrow Aspirate (collected
- Pathologist
from iliac crest; assist the hematologists - Additional Test
to put the sample in the tubes; bone - Specimen Label
marrow is fast to clot; less than 3 - Gross Examination
seconds it must be mixed with anti- - Diagnosis
coagulant)
- Write the important details of the patient.
o Accession number
o Patient’s name
o Age & Sex
o Room number
o Attending Physician
o Specimen
o Transaction number
o Patho-on-deck
o Date received / Due Date.
- SPECIMEN ACCESSIONING FORMAT
o SP-20-143-A
 SP: Type of Specimen
 AP: Autopsy Pathology
 SP: Surgical Pathology
 20: Last 2 digit of the year 6. KEEP ALL DOCUMENTS IN ONE FOLDER
 2019 = 19

JLLD 3
Histopathology and Laboratory Management

- Folder is for processing sample: meaning it is  Received in one part in 10%

done in gross examination. neutral buffer formalin filled
- Follow ups from doctors can be answered container
easily through this folder especially when it is  One part = one
in the processing tray. container
- For signing: results are already typed and  Unlabeled: quote unlabeled
must be signed by the patho, the result o Gross Description
cannot be released unless signed. o Microscopic Description
- This folder can be used for other tests as well. o Diagnosis
o Name and Signature of Pathologist
RELEASING OF RESULTS o Transcribed by: Date and Time
- Signed result for releasing
- Check for additional tests. o It is recommended to place the final
- Don’t forget to read endorsement letters from
result in an envelope to maintain patient
your colleagues.
confidentiality.
o Additional work-up must be
- Releasing logbook
accommodated o Date today/ Date released.
o Why is IHC requested?
o Name of the patient
 ER/PR Her2neu
o Name and sign of the person who
 Estrogen and Progesterone
her2-neu assay claimed the result.

Malignant or cancerous diagnosis ang patient it must be GROSS EXAMINATION, FIXATION,

treated, so if ER is positive doon mag focus ang clinician DEHYDRATION, AND DECALCIFICATION
sa Estrogen receptor, doon ang focus ng treatment
 If PR is positive, then it will be GROSS EXAMINATION
focused o n progesterone - A knowledge of what needs to be taken for
receptor and doon ang focus ng microscopic is crucial for final diagnosis
treatment - Crucial: bc kung ano yung masubmit na
 Her2neu: this is will be hard specimen yun na rin ang ma examine under
because recurrence incidence is microscope – so dapat yung mga na cut or
likely to happen, the clinicians submit na specimen for tse processing is
approached in treatment is enough for them to arrive to a proper diagnosis
through adjuvant, aside from - IMPORTANT STAGE IN HISTOPATHOLOGY
chemotherapy is radiation - Examined by
therapy since it can be o Pathologist
transferred to another. o Pathology Assistant or
 Triple negative is even more o Pathology Resident
hard bc the doctor cannot give a o Sometimes this is assigned to Medtech
treatment, or the doctor will ask - It involves:
to proceed for FISH. o Accurate naked eye description of intact
- Double Check Everything specimen
o BETWEEN Surgical Path Worksheet  Describe the outer appearance
and Final Biopsy Result (e.g. lisa rosa – smooth) they
- “For Further Evaluation” Cases will also measure it in 3d (l*w*h)
o These are cases wherein the patho-on- o Correct method of sectioning
deck prefers to have a a consultation or o Gross examination of cut surface
refer the case to other pathologists.  Cut surface is described from
- Sample Results: what was seen inside when it
o Name was cut (e.g. cut section
o Address shows…)
o Physician o Selection of proper tissue blocks for
o Procedure performed microscopy
o Specimen  Dapat ang ma submit na
sample is enough to diagnose

JLLD 4
Histopathology and Laboratory Management

o Instructions for embedding and block o 0.5% acetic acid solution is used as
making fixative
 Pathologists will also instruct  Fixative for the ink
the histotech paano and ano o E.g. breast is submitted in lab to
ang gusto niya i-pa expose examine if to what extent nag abot ang
 Orientation of tissue defines the tumor cells
plane of section in embedding - Surgical margins
- Crucial in clinical management and provide o Lateral
important prognostic data o Medial
o Superior
GROSSING AREA
o Inferior
- Grossing stations allow gross examination of o Basal
surgical specimens which are typically
o Border where the surgeon cut
performed by a pathologist, or by a pathologists’
 Tested for the positive or
assistant working within a pathology practice.
negative as a tumor cell
o Maraming gamit
 Ideally dapat negative kasi it
o Gripo: formalin
means high chance na nakuha
o Inks: inking (for margin) na lahat ang tumor cells
o Scissors - Ink is for label of what margin
o Guide
o They sometimes self-record and say GENERAL PRINCIPLE OF GROSS EXAMINATION
what they see and the medtech will later 1. Specimen Identification
transcribe it a. Doon sa sample result makita ang
- Instruments received in one-part neutral buffered
o Gloves formalin and labeled as…
o Formalin 2. Identify all the anatomical structures present
o Blade – mas prefer sa doctors mag use a. E.g. appendix: fibro fatty tissue
ng blade when they are going to set the 3. Orientation markers should be identified
size of the sample to be put in the a. Possible lang ito siya sa malalaking
cassette (2x2 cm) and the specimen specimens for marking and identification
must fit into the cassette 4. Measurements: Length width height (3D)
o Ruler – the pathologist will measure the 5. Weight: especially parenchymatous organ
mass and the specimen (Prostate chips)
o Forceps 6. Examine the external surface
o Cassette a. Smooth, rough, serosa, what you see is
o Cutting board / Chopping Board what you describe
7. Cut all the organs at intervals of 1 cm thickness
o Large Scissor – used in tubular
a. Bread loafing
specimens like intestines
8. Describe cut surface, identity pathologic process
o Scalpel – alternative for blades
9. If suspected lesion is present, measure,
o Dissecting scissors – for small areas or describe with the color and consistency
specimens (e.g., lymph node) a. Separate measurement for the lesion
o Saw – for hard specimens (e.g., identified
amputated leg) 10. Surgical Margins
11. Histological section
INKING
- When working in histopathology as a surgical SMALL BIOPSIES
crossing tech you have to learn a new language When will we work on this? How much time do we need
and this language must be clear and concise so to execute this?
there are a few terms that you may see
(e.g., endometrial curettage and skin punch biopsy,
o Flat pigmented lesion = macule
“specimen consists of tan to black fragments of tissue
- Madaming new terms from the doctors
aggregately measuring 0.5 cm -one dimension)
- Histopathological evaluation of surgical margins
of a resected tumor specimen can give an 1. Number of fragments, aggregate dimension
insight about the extent of tumor spread 2. Greatest dimension of largest fragment
3. Shape of fragment, Color and Consistency

JLLD 5
Histopathology and Laboratory Management

a. Tan to brown soft irregular piece of - The broad objective of tissue fixation is to
tissue (piece of tissue = one) preserve cells and tissue components in a “life-
4. Supported within the gauze or filter paper to like state” and to do this in such a way as to
prevent tissue loss during processing allow for the preparation of thin, stained
5. All fragments are submitted sections.
6. “Submitted in one block” “block 2” (depends on o From the time na nakuha ng surgeon
the cassette; 2 cassette = 2 block) ang specimen nalagay na agad sa
APPENDIX fixative to stop autolysis
 Autolysis: results in tissue
Procedure digestion by intracellular
1. Measure organ (length and greatest diameter) enzymes released when
2. Divide specimen in two by cutting a cross- organelle membrane rupture
section 2 cm from tip  Bacterial decomposition or
3. Cut cross-sections of proximal fragments at 5 putrefaction brought about by
mm intervals the microorganism
4. Divide distal fragments in two by a longitudinal - For practical purposes fixation aims to prevent
cut or arrest the degenerative processes which
5. Description commence as soon as a tissue is deprived of its
a. L*W*H blood supply
b. External surface o Loss & diffusion of soluble substances
c. Perforation must be avoided as far as possible
d. Wall Thickness - Tissue processor:
e. Mucosa
f. Lumen
g. Content
“Specimen consists of tan to red appendix measuring
4x3x1 cm, the external surface is smooth, cut section
shows a dilated lumen with a fecaloid material, wall
thickness is 0.1 cm. block 2”
- Section for histology
- Proximal 1/3 close to surgical margin; one cross-
section
- Mid 1/3: one cross section
- Distal 1/3: one longitudinal section
- If tumor is present, perform inking and take an
additional section from it
- Tissue Basket: kung saan ilalagay ang cassettes

- Basket is hanged in the processor, and it is the

processor itself that will dip/immerse it to the
FIXATION reagents
o Formalin

JLLD 6
Histopathology and Laboratory Management

o Ascending grades of alcohol

o Xylene
o Wax
- Tissues must be largely protected against the
deteriorating effects of the tissue processing
including infiltration with hot wax

GENERAL PPRINCIPLES OF FIXATION

1. First and most critical step
2. Cell and tissue constituents are preserved with
the least alteration from the living state with the
use of fixatives
3. Prevents the breaking down of cellular elements
via autolysis by inactivating lysosomal enzymes, FACTORS AFFECTING FIXATION
making the tissue components insoluble 1. pH
4. Must also render the specimen insensitive to o ideal ph is 6-8, for ultrastructure
subsequent treatment as may be necessary to preservation it is buffered between 7.2-
permit microscopic examination 7.4 so the 10% formalin will react most
- Tissues must retain reactivity to stains and other effectively at neutral ph
reagents including antibodies and nucleic acid o usually, ma buffer siya up to 6.8 to 7.2
- It is important to realize that fixative will initially o unbuffered formalin (e.g., concentrated
produce a number of changes to the tissues in
formalin) it will slowly be oxidized to
what is usually an aqueous environment
formic acid resulting in a fall in pH
- This will include
o formic acid will also react with
o Shrinkage
hemoglobin forming formaldehyde
o Swelling
hematin
o Hardening  hematin: brown-black granular
o Of various components artifact pigment which is
- E.g. fixation is carried out in 10% buffered deposited in blood rich tissues
formalin bc it causes slight swelling of tissue  nuisance as it can be confused
specimens with other microorganisms or
**Paraffin section of kidney that has been fixed using other pathological fragments
neutral buffered formalin. This is an example of well-  it can be removed by saturated
fixed tissue showing good nuclear and cytoplasmic picric acid but ofcourse
morphology with minimal shrinkage showing clearly prevention is better
defined basement membranes and cell margins. 2. Osmolality
o Osmotic effects that are more important
at the ultra-structural level than at light
microscopy level
o It is the phospholipid membranes that
are easily damaged by excessive hypo
or hypertonic solutions
o Before fixation cells can be certainly
damaged by non-isotonic fluids such as
water
o So if specimens cannot be fixed
immediately they can be kept moist with
gauze soaked in isotonic saline for a
**Paraffin section of kidney that has been fixed using a short time
neutral buffered formalin. This is an example of poorly- o It is not a good idea to hold tissue
fixed tissue showing inferior nuclear and cytoplasmic immersed in saline for extended period
morphology with excessive shrinkage and poorly defined o Pasa agad sa histopath asap if nasa
cell margins. Note the vacuolation and fragmentation of isotonic saline lang
both nucleus and cytoplasm of cells of the distal tubule 3. Size of the Specimen
and retraction of the glomerulus due to shrinkage. o Not more than 4 mm thick

JLLD 7
Histopathology and Laboratory Management

o Ideally 3 mm thick should provide - Non coagulant fixatives which are additive in
excellent fixation and processing nature formed extensive crosslinks producing a
o Penetration of formalin to the tissue is 1 less permeable gel
mm per hour o Still encountered in a modern
o So the bigger the specimen is mas hindi histological literature
na makapenetrate ang formalin
o Bigger specimen = longer fixation DENATURATION
process - Most commonly this effect is induced by
4. Volume of the fixative dehydrants such as the alcohols or acetone.
o 20:1 Fixative to tissue ratio These reagents remove and replace free water
o It is important to have an excessive in cells and tissues and cause a change in the
volume of fixative in relation to the tertiary structure of proteins by destabilizing
volume or to the total volume of tissue hydrophobic bonding.
o Because with additive fixatives the o Hydrophobic areas frequently found on
effective concentration of reagent is the inside of protein molecules and are
depleted as fixation proceeds released from the repulsion of water and
o In a small total volume this could have become free to occupy a greater area
o Hydrophilic areas well mixed with water
an effect on fixation quality
5. Temperature are loosely bound by hydrogen bonds
o Higher temperature = higher rate of and removal of water destabilizes these
bonds
diffusion = higher rate of chemical
o So the changes produce in the
reaction between the fixative and tissue
elements conformation of the protein molecules
o It can also increase the rate of tissue cause a change in the solubility of the
protein
degeneration in unfixed areas of tissue
o So rendering water soluble proteins in
o Microwave fixation may involve higher
soluble
temperature up to 65 degrees Celsius
o So a change that is largely irreversible
but for relatively short periods only
6. Time is the protein is returned to an aqueous
o Time varies depending on the fixatives environment
o Fixative has to penetrate by diffusion to **a paraffin section from the mucosa of small intestine
the center of the specimen and that has been fixed in 95% ethanol, a denaturing
sufficient time has to be allowed for the fixative. While nuclear presentation is fair there is a
reactions of the fixation to occur substantial shrinkage of cytoplasmic and extracellular
o FORMALIN = 24 hours elements.
o Pre-fixed tissue that is received is
processed the next day so that it is well
fixed for processing (minsan sobra pa
24 hours)
7. Penetration rate
o 1 mm per hour
o Depends on diffusion characteristics
o Varies from one reagent to another

CLASSIFICATION OF FIXING AGENTS AND THE

MECHANISMS OF FIXATION
- Traditionally fixing agents were termed - The non-coagulant fixing agents chemically
“coagulant”, or “non-coagulant” based on their react with proteins and other cell and tissue
effect on soluble proteins in solution components, becoming bound to them by
- There are two major mechanisms which are addition and forming inter-molecular and intra-
important in fixation of proteins and proteins molecular cross-links. Because these agents are
complexes; denaturation, and addition and reactive compounds they bind to a variety of
cross-link formation chemical groups in tissues, often affecting the
- Coagulant fixatives were said to result in a charge at the site of attachment
permeable mesh work of protein strands

JLLD 8
Histopathology and Laboratory Management

- Tissue fixed with formaldehyde stains poorly o Ethanol: most widely used dehydrant, is
with eosin because formaldehyde reacts a drinking alcohol and hence strictly
extensively with amino groups and forms controlled
methylene bridges and thus these groups are no o Methanol
longer available to bind negatively charged dye o Isopropanol
molecules such as those of eosin o Butanol
- The extent to which additive fixatives form - Reagent: Denatured Alcohol Ethanol
cross-links vary considerably - 70%-95%-100% gradually increasing
o E.g., glutaraldehyde is more effective at concentration
forming crosslinks than formaldehyde
DEHYDRATING AGENT IDEAL
o Explains why glutaraldehyde effectively CHARACTERISTICS
preserves the ultrastructure of cells and 1. Dehydrates rapidly without producing
is the fixative of choice for electron considerable tissue damage
microscopy 2. Not evaporate too fast
3. Not harden tissue excessively
**a paraffin section of the mucosa of small intestine that 4. Not remove stains
has been fixed in neutral buffered formalin, a cross- 5. Not a fire hazard
linking fixative. Nuclear and cytoplasmic preservation is 6. Not toxic to handler
satisfactory, but some cellular shrinkage is present. 7. Able to dehydrate a wide range of tissue

FACTORS AFFECTING DEHYDRATION

1. Agitation – mechanical; increase the speed in
which the alcohol replaces the water (machine
moves up and down for agitation)
2. Heat – permits gentle heat to be applied during
dehydration; since warming fluid makes them
less viscous it increases the effectiveness of
dehydration by increasing the ability to penetrate
3. Time – long slow dehydration gives the best
result (overnight processing is most common)
4. Shrinkage – done in room temp starting with
moderate concentrations of alcohol causes a
little tissue shrinkage
DEHYDRATION
DIFFERENT DEHYDRANTS
- Tissue basket will emerge on to 70% alcohol
then move to 95% and then another two 1. Alcohol
absolute alcohol a. Ethanol
b. Methanol
GENERAL PRINCPLES OF DEHYDRATION c. Isopropanol
1. Removal of water before infiltration 2. Glycol-Ethers
o Melted paraffin wax is hydrophobic a. 2-Ethoxyethanol/ Ethylene Glycol
Monoethyl Ether (Cellosolve)
2. Immersing the specimen in a series of
b. Dioxane
increasing concentrations of alcohol
c. PEGS – Polyethylene glycols
o Ultimate concentration is pure, water-
free alcohol (absolute) ETHANOL
o Mag-shrink and mag brittle ang - In absolute ethanol
specimen if direct to absolute alcohol o minimal processing time
3. Minimize contact time with chemicals limits o progressive removal of bound water
tissue distortion from proteins CHON and carbohydrates
4. Minimize risk of extracting cellular components CHO
5. Ideal fluid to tissue ratio = 10:1 o during prolonged immersion tissue
harden excessively and become brittle
PROCESSING REAGENTS: DEHYDRANTS
o ideal for delicate tissue
- Most dehydrating reagents are alcohols
METHANOL

JLLD 9
Histopathology and Laboratory Management

- good ethanol substitute but rarely used for 1. Dissolution of calcium by a diluted mineral ACID
routine processing 2. Using of Chelating Agents
- volatile, flammable, costly - Most common chelating agent: EDTA (pH:
- poor lipid solvent 7)
- does not dissolve nitrocellulose (unless mixed - Function: Chelates calcium to form a soluble
with acetone) salt.
ISOPROPANOL - Forms: VERSENE (SOLID) and
SEQUESTRENE (LIQUID)
- universal solvent 3. Removal of calcium by using a diluted mineral
- slightly slower in action and not as hygroscopic and along with ION EXCHANGE RESIN to keep
as ethanol the decalcifying fluid free of calcium.
- superior lipid solvent 4. Electrolytic removal of calcium ions from tissue
- since specimens submitted are not usually fatty by the use of electric current:
tissues ethanol is still most preferred ELECTROPHORESIS
5. Microwave oven decalcification – faster than the
routine procedure (inc tissue damage)
STRONG ACIDS

A. NITRIC ACID
- COMPOSITION
o Concentrated Nitric Acid: 10 mL
o Distilled Water: 100 mL
- ADVANTAGES
o Rapid in action
o Produces minimum shrinkage
o Produces good nuclear staining
DECALCIFICATION o Recommended for urgent biopsy,
- REMOVAL OF CALCIUM ions from a bone or needle and small biopsy
calcified tissue through a histological process - DISADVANTAGES
that makes them flexible and easier to cut. o Prolonged decalcification may lead to
tissue distortion
PATHOLOGIC CALCIFICATION o Damage tissue stainability
- Abnormal tissue deposition of Calcium salts o Imparts a yellow color with nitrous acid
- Smaller amounts of Fe, Mg, & other mineral o Strong acids tend to be more damaging
salts to tissue antigens (histochemical
- DYSTROPHIC – deposition occurs locally in staining)
dying tissues
- METASTATIC – deposition in normal tissues
B. PERENYI’S FLUID
DECALCIFYING IS AFFECTED BY THE - COMPOSITION
FOLLOWING: o 10% Nitric Acid: 40 mL

JLLD 10
Histopathology and Laboratory Management

o 0.5% Chromic Acid: 30 mL

o Absolute Ethanol: 30 mL
- ADVANTAGES
o Recommended for routine purposes
o Decalcifies and softens tissues at the
same time
o Nuclear and Cytoplasmic staining is
good
o Maceration is avoided due to the
presence of chromic acid and alcohol
- DISADVANTAGES
o Slow decalcifying agent for dense bones
o Complete decalcification cannot be
determined by chemical test

C. PHLOROGLUCIN-NITRIC ACID
- COMPOSITION
o Concentrated Nitric Acid: 10 mL
A. FORMIC ACID
o Phloroglucin: 1 gm - COMPOSITION
o 10% Nitric Acid: 100 mL o Formic acid: 10 mL
- ADVANTAGES o 10% Normal saline: 90 mL
o Most rapid decalcifying agent - ADVANTAGES
o Recommended for urgent cases o Both a fixative and decalcifying agent
- DISADVANTAGES o Permits excellent nuclear and
o Nuclear staining is poor cytoplasmic staining
o Prolonged decalcification produces o Recommended for small pieces of
extreme tissue distortion bones and teeth
o Yellow color must be neutralized with o Suitable for most routine surgical
5% sodium sulphate and thoroughly specimens
washed with running tap - DISADVANTAGES
o water (24 hours) o Relatively slow, not suitable for urgent
o Complete decalcification cannot be specimens
determined by chemical means o Requires neutralization with 5% sodium
sulfate
D. VON EBNER’S FLUID
- COMPOSITION B. TRICHLOROACETIC ACID
o Saturated aqueous solution of NaCl: 50 - COMPOSITION
mL o Trichloroacetic acid: 5 gm
o 36% Concentrated HCl: 8 mL o 10% Formal saline: 95 mL
o Distilled Water: 50 mL - ADVANTAGES
- ADVANTAGES o Permits good nuclear and cytoplasmic
o Permits good cytologic staining staining
o Moderately rapid decalcifying agent o Does not require washing out
o Does not require washing - DISADVANTAGES
o Recommended for teeth and small o Weak decalcifying agent, not used for
pieces of bone dense tissues
- DISADVANTAGES o Very slow-acting, not recommended for
o Extent of decalcification cannot be urgent examinations
measured by chemical test
WEAK ACID C. CHROMIC ACID
- COMPOSITION

JLLD 11
Histopathology and Laboratory Management

o Chromic acid: 15 mL facilitate solution, ionization and removal

o Osmium Tetroxide: 4 mL of calcium.)
o 2% Glacial Acetic Acid: 1 mL 3. SIZE AND CONSISTENCY
- ADVANTAGES a. Increase in size and consistency will
o Both fixative and decalcifying agent require longer periods for complete
o Used for decalcifying minute bone decalcification.
spicules b. Ideal: 24-48 hours
- DISADVANTAGES c. Dense tse: 2 weeks or longer
o Nuclear staining with hematoxylin is 4. AGITATION
a. Gentle agitation may increase the rate
inhibited
of decalcification. It is achieved by low-
o Tends to undergo reduction and forms
speed rotation, rocking, stirring or
precipitates at the bottom of container
bubbling air into the solution.
o Insoluble pigments are formed when
b. Slowly dislodge precipitates from tse
dehydrated with alcohol 5. TEMPERATURE
o Degree of decalcification cannot be a. Increase temperature will hasten
measured by the routine chemical test decalcification, but it will also increase
the damaging effects of acids on tissue.
b. Heat hasten decalcification
c. Lower temp = decrease reaction rate

D. CITRIC ACID-CITRATE BUFFER SOLUTION WAYS OF MEASURING THE EXTEND OF

- COMPOSITION DECALCIFICATION
o 7% Aqueous solution Citric acid
There are several methods to check if the end point of
(monohydrate): 5 mL decalcification has been reached:
o 7.4% Aqueous solution Ammonium
Citrate (anhydrous): 95 mL 1. PHYSICAL/MECHANICAL
o 1% Aqueous solution Zinc Sulfate: 0.2 o inaccurate a.k.a. FLEXIBILITY
mL METHOD
- ADVANTAGES o folding/bending
o Permits excellent nuclear and 2. X-RAY/RADIOLOGIC METHOD
cytoplasmic staining o very expensive; most reliable; most
o Does not produce cell or tissue ideal
distortion o checks thru x-ray
- DISADVANTAGES 3. CHEMICAL METHOD/CALCIUM OXALATE
o Action is too slow for routine purposes TEST
o simple, reliable, recommended for
routine purposes
o can’t be used if the acid used is
FACTORS INFLUENCING THE RATE OF
DECALCIFICATION Perenyi’s, Von Ebner, & Phloroglucin-
Nitric Acid
The rate of decalcification may be influenced by several
factors TISSUE SOFTENER
1. CONCENTRATION For unduly hard tissue that may damage the microtome
a. more concentrated acid solutions knives
decalcify bone more rapidly but are
- 4% Aqeous Phenol (1-3 hours)
more harmful to the tissue.
- Molliflex – soapy appearance
b. Concentration of active ingredient and
- 2% HCl
volume of decalcification agent is
- 1% HCl in 70% Alcohol
depleted as it combines with calcium
- Perenyi’s Fluid
2. FLUID ACCESS
a. a fresh decalcified should have ready POST-DECALCIFICATION
access to all surfaces of the specimen
(enhance diffusion and penetration and

JLLD 12
Histopathology and Laboratory Management

Removal of excess acid is done by immersing the o (Miscible-is the ability of a substance to be
decalcified tissue in: mixed well Solubility-is the ability of the
- Saturated Lithium carbonate substance to be dissolved)
- 5- 10% Aqeous Sodium bicarbonate o (Clearing is the bridge between Dehydration
and infiltration that why it is one of the
COMMON PRACTICE:
crucial steps)
- Rinse the decalcified specimens with running o (Note: In H.E STAINING the first 3 clearing
tap water, agents are for the final deparaffinization.
o (In PAPSMEAR when used after tissue has
been stained, the clearing agent will make
microscopic tissue preparation transparent
due to their high index of refraction)
 Since most alcohols and paraffin are NOT miscible,
another step, known as clearing, is introduced.
 After the dehydrating agent is removed, the tissue will
have a translucent appearance = thus the procedure
is called “clearing”
o The clearing of the tissue is due to the
increase in the refractive index of the tissue
caused by the clearing agents
 THE PURPOSE OF THE CLEARING IS TO
FACILITATE THE PENETRATION OF THE
TISSUE BY THE EMBEDDING MEDIUM WHICH
IS THE WAX

CHARACTERISTICS OF A GOOD CLEARING

NOTES TO REMEMBER AGENT
1. more concentrated acid solutions decalcify 1 Should be miscible with alcohol to promote rapid
bone more rapidly but more harmful to the removal of the dehydrating agent
tissue 2 Should be miscible or is easily removed by the
2. high concentrations and greater amount of melted paraffin wax to facilitate impregnation of
fluid will increase the speed of the process. the tissue
3. heat will serve to hasten decalcification, but 3 Should not produce excessive tissue shrinkage,
it also increases the damaging effects on hardening and damage
tissues. 4 Should not dissolve dyes
4. the ideal time required for decalcifying 5 Should not evaporate quickly in water bath but
tissue is 24-48 hours. should readily evaporate in the oven
5. dense bone tissue usually requires up to 14 6 Should make tissue transparent
days or longer to complete the process.  Recommended volume: 10x the tissue
sample

Take note: Most clearing agents are flammable

liquids that warrant considerable caution in their
use.

XYLENE
CLEARING “DEALCOHOLIZATION”  30 mins to 1 hour
 Most commonly used clearing agent in routine
 Process whereby alcohol or a dehydrating agent is biopsies
removed by the tissue and replaced it with a  Used for clearing, both embedding and mounting
substance that will dissolve with the wax that will be procedures
used in impregnation
 Colorless chemical and inexpensive
 Process of replacing the dehydrating fluid with a fluid
that is miscible with both the dehydrating fluid and ADVANTAGES
impregnation or embedding medium

JLLD 13
Histopathology and Laboratory Management

 CLEARING TIME: 15-30 MINS (MOST RAPID  At very high levels of exposure, xylene can injure
CLEARING AGENT) the liver and kidneys.
 Miscible with alcohol and paraffin
 Makes tissue transparent BENZENE
 Does not affect staining  RAPID ACTING: CLEARING TIME: 15-60MINS
 Evaporates rapidly in the oven thus can easily be  Also recommended for urgent biopsies
replaced by the melted paraffin  Same as xylene, highly volatile inside the oven
DISADAVATANGES thus is easily replaced by paraffin wax
 Miscible in alcohol and makes tissue transparent
 Highly flammable  Unlike xylene, it is preferred by some because it
 Tissue immersed in xylene for more than 3hrs doesn’t cause tissue to become hard and brittle
become hard and brittle  If tissue is immersed in benzene for a longer
 Can cause tissue shrinkage thus not period of time, there is considerable tissue
recommended for biopsies of lymph nodes and shrinkage
nerves  Chronic exposure to Benzene can cause bone
 Becomes milky if there is still water in the tissue marrow suppression resulting to aplastic anemia
NOTE:  Benzene is also a carcinogenic – acute myeloid
 Make sure dehydration process is done leukemia
completely o Aplastic anemia
 Repeat dehydration using absolute alcohol o Acute myeloid leukemia
 Exposure to high levels of xylene can irritate the  Exposure to benzene may be harmful to the
lungs, causing chest pain and shortness of breath. reproductive organs.
 Extreme overexposure (e.g., in a confined space)  Some women workers who breathed high levels
can result in pulmonary edema, a potentially life- of benzene for many months had irregular
threatening condition in which the lungs fill with menstrual periods. When examined, these
fluid. women showed a decrease in the size of their
 Its vapor is heavier than air and may accumulate ovaries.
in low-lying areas.
 Children exposed to the same levels of xylene TOLUENE
vapor as adults may receive a larger dose  CLEARING TIME: 1-2HRS
because they have greater lung surface area:  Used as substitute for xylene and benzene
body weight ratios.  Recommended for routine purposes
 In addition, they may be exposed to higher levels ADVANTAGES:
than adults in the same location because of their
short stature and the higher levels of xylene vapor  Even if tissue is left for 24hrs in toluene, there is
found nearer to the ground. no shrinkage, brittleness and hardening
 Inhalation of xylene vapors in small amounts can  NOT carcinogenic unlike Benzene
cause headache, dizziness, drowsiness, and DISADVANTAGE
nausea.  Slower than xylene and is EXPENSIVE
 Effect of xylene on the central nervous system is  Emits fume that can be toxic to humans
attributed to the liposolubility of xylene in the  CNS effects (headache, dizziness, drowsiness,
neuronal membrane.
euphoria, hallucinations, tremor, and seizures)
 It has been suggested that xylene disturbs the o Liver and kidney damage
action of proteins essential to normal neuronal
o Cardiac arrythmia
function either by disruption of the lipid
environment in which the membrane protein’s  Breathing high levels of toluene during
function or by direct interaction with the proteins in pregnancy has been shown to result in children
the membranes. with birth defects and to retard mental abilities
and growth.
 Xylene vapor is only mildly irritating to mucous
membranes; however, xylene splashed in the  There is evidence that exposure to toluene at
eyes can result in corneal injury. work is associated with spontaneous abortion.
 Repeated or prolonged skin contact with liquid
xylene can defat the skin, causing it to crack and
peel (it will dissolve the natural oils in the skin) CHLOROFORM
 CLEARING TIME: 6-24HRS

JLLD 14
Histopathology and Laboratory Management

ADVANTAGE  Celloidin is another chemical used in embedding

 NOT FLAMMABLE  CLEARING TIME: 2-3 DAYS
 Can penetrate even thick tissue sections and  Works well with celloidin and tissues are easy to
large tissue specimen cut
 Does not cause tissue shrinkage that’s why its  Minimal tissue hardening and shrinkage
ideal for biopsies of lymph nodes and nerves  Slow clearing agent thus not recommended for
 ideal for skin, fibroid tissue, decalcified tissue urgent biopsies
 Hard to eliminate from tissues in paraffin oven
DISADVANTAGE thus impregnation with parafiin would require
 Does not make tissue transparent additional time
 Not very soluble in paraffin and does not easily  Tissues placed in cedarwood floats before they
evaporate in the oven thus impregnation will gradually settle at the bottom
take time  Superficial portion of tissue may dry out before it
 Tissues float in chloroform because it has a is cleared
bigger density thus affecting the clearing  Upon storage, cedarwood oil becomes milky and
process requires filtration before use
o Wrap tissue with cotton gauze to make it  Very expensive
settle at the bottom of the container  If the fixative used is acetic alcohol, cedarwood
 Toxic to the central nervous system and liver after oil forms crystals which are removed by heating
prolonged inhalation the cedarwood oil at 200 degree Celsius if you
o Lower concentrations (resulting in dizziness, still wish to use it for other tissues
headache, tiredness)
o At very high levels, chloroform exposure ANILINE OIL
may result in death. - Recommended for small and delicate tissues
 Chronic exposure to chloroform in humans is because it does not cause excessive tissue
associated with effects on the liver, including shrinkages and hardening
hepatitis. - Embryos, insects, and very delicate specimens
 Reproductive effects, such as decreased conception
rates, decreased ability to maintain pregnancy, and METHYL BENZOATE AND METHYL SALICYLATE
an increase in the percentage of abnormal sperm  Slow-acting
were observed in animals exposed to chloroform  Can be used when double embedding
through inhalation. techniques are required
 Animal studies have noted decreased fetal weight, IMPORTANT THINGS TO TAKE NOTE OF:
increased fetal resorptions, but no evidence of birth
defects, in animals orally exposed to chloroform.  Boiling point of clearing agent
o The lower the boiling agent, the faster it
CARBON TETRACHLORIDE is replaced by paraffin
 Properties and disadvantages are similar to o Xylene has a low boiling point while
chloroform chloroform has a higher boiling point
 Can cause minimal tissue hardening (that’s why it’s not easily replaced by
 Cheaper than chloroform paraffin)
 Human symptoms of acute (short-term)  Viscosity
inhalation and oral exposures to carbon o The lower the boiling agent, the faster it
tetrachloride include headache, weakness, is replaced by paraffin
lethargy, nausea, and vomiting. o Xylene has a low boiling point while
 Acute exposures to higher levels and chronic chloroform has a higher boiling point
(long-term) inhalation or oral exposure to carbon (that’s why it’s not easily replaced by
tetrachloride produces liver in humans. paraffin)

CEDARWOOD OIL CHOICE OF CLEARING AGENTS DEPENDS ON:

 Can be used in tissues to be embedded using  Type of tissue
paraffin or celloidin  Type of processing and processor system
o (Recommended for CNS tissues and
 Intended processing conditions (temperature,
cytological studies commonly example vacuum, pressure)
is smooth muscle and skin)

JLLD 15
Histopathology and Laboratory Management

 Safety factor, cost and convenience

IMPREGNATION AND EMBEDDING

 The tissues, after fixation, dehydration and
clearing process, are not sufficiently hard to cut
into thin sections without a suitable support.
IMPREGNATION/INFILTRATION  Process is very rapid, allowing sections to be
 Process whereby the clearing agent is completely prepared within 24 hours (from dehydration to
removed from the tissue and is replaced by a staining
medium that will completely fill all the tissue cavities,  Ideal for routine tissue processing
 Giving a firm consistency to the specimen  Tissue blocks and unstained mounted tissue
 allowing easier handling and cutting of suitably thin sections may be stored in paraffin in an
sections without any damage to the tissue and its indefinite period of time after impregnation
components without considerable tissue distortion
 (The medium used to infiltrate the tissue is usually  Many staining procedures are permitted with
the same medium used for embedding) good results

TISSUE BLOCKS FOR STORAGE

4 GENERAL TYPES OF TISSUE IMPREGNATION  Overheated paraffin can cause tissue to shrink
AND EMBEDDING MEDIA and to become brittle
1 Paraffin wax o Pag masyado siyang HOT, nagiging
2 Celloidin MARUPOK ka
o Lymph nodes and nerves are lost
3 Gelatin o Maintain temperature to 2-5 degree
4 Plastic/Resin above melting point of wax
 Prolonged impregnation will cause excessive
PARAFFIN WAX tissue shrinkage and hardening, making the
 Simplest, most common and best embedding sections difficult to cut
medium used for routine tissue processing  Inadequate impregnation will cause the retention
 Thin sections (4-6 um) may be cut with east of clearing agent
from majority of tissues without distortion o Tissue becomes soft and shrunken
o (Not recommended for FAT specimen)
o (Paraffin oven/Incubation temp: 55-60C) o Tissue blocks crumble or break up when
o (Paraffin oven MUST maintain a floated in a water bath
temperature 2-5C ABOVE MELTING Complete Infiltration Tissue blocks crumble
POINT of the paraffin wax)
 Paraffin is relatively hard thus sections can
easily be cut
 Serial sectioning is easy to do
o (Note: Tissues difficult to infiltrate
(Bones, teeth, brain, and eyes) -need
long proper support. And not
recommended for fatty tissues) TISSUE BLOCKS BREAK UP WHEN FLOATED IN
A WATER BATH
SERIAL SECTIONING  Paraffin processing is not recommended for fatty
 Serial sectioning is defined as obtaining a tissues.
continuous ribbon of sections from a paraffin  The dehydrating and clearing agents used in the
block and placing all the sections on multiple process dissolve and remove fat from the tissue
slides  Take note: the medium used in infiltration is the
same chemical used in embedding
 During infiltration, the medium should be in liquid
state.
 During embedding, the medium should solidify

JLLD 16
Histopathology and Laboratory Management

 To keep the medium in liquid state during  At least 4 changes of wax are required at 15
infiltration, the medium should be in melted. minutes intervals in order to insure complete
 Higher melting point means the wax is difficult to removal of clearing agent from the tissue.
melt and therefore, it is a hard type of wax!  The specimen is then immersed in another
 Conclusion: The higher melting point, the harder fresh solution of melted paraffin for
the wax…. approximately 3 hours to insure
 The harder the wax, the easier it is to make thin completeness of embedding or casting
sections...
AUTOMATIC PROCESSING
 Hard wax requires heavy duty type of microtome
during sectioning!!!  Makes use of an automatic tissue processing

PARAFFIN WAX
• commonly available paraffin waxes have the ff
melting point in:
o 45 degree Celsius
o 52 degree Celsius
o 56 degree Celsius – most commonly
used
o 58 degree Celsius
 The recommended temperature for infiltration is
usually 2-5 degree Celsius above the melting machine which fixes, dehydrates, clears and
point of the wax to ensure it is completely infiltrates tissues thereby decreasing the time
melted. and labor needed during the processing of
 56-degree Celsius wax = Temp 58-61 tissues resulting to rapid diagnosis with less
 58-degree Celsius wax = Temp: 60-63 technical errors
o Higher temperatures can tissue to Oscillating tissue basket
become brittle and shrunken and these • The tissue basket oscillates up and down in
changes will start at temperatures above each station at three-second intervals to ensure
60 degree Celsius thorough and even mixing of the reagents and
optimum tissue infiltration

PROCEDURE 10 BEAKERS OR JARS

 After clearing, the tissue is submerged in 2-4 • Transparent beakers
changes of melted paraffin wax, either in a • Beaker I – fixative (formalin) 1-2 hours
paraffin oven or in an incubator regulated at 55-
60 degree Celsius • Beaker II – fixative 1 hour

 In a room temperature (20-24 degree Celsius), • Beaker III – fixative. 30- 45 minutes
paraffin wax with a melting point of 54-58 degree • Beaker IV – 70% alcohol. 30 minutes
Celsius is recommended • Beaker V – 90% alcohol. 30 minutes
 If the laboratory temperature is between 15-18 • Transparent beakers
degree Celsius, the melting point of the wax to
• Beaker VI – Absolute alcohol. 1 hour
be used should be between 50 - 54 degree
Celsius • Beaker VII – Absolute alcohol. 1 hour
• Beaker VIII – Absolute alcohol. 1 hour
THREE WAYS OF PARAFFIN WAX
• Beaker IX – Xylene. 1-2 hours
IMPREGNATION AND EMBEDDING
1 By manual processing • Beaker X – Xylene 45 minutes – 1 hour
2 By automatic processing • Thermostatically controlled beakers
3 By vacuum embedding • Wax bath I (done at 45°c) 2 hours
MANUAL PROCESSING • Wax bath II. 2 hours

JLLD 17
Histopathology and Laboratory Management

When it’s time for tissue to be transferred to the next  Involves the wax impregnation under negative
beaker or jar, the cover of the machine is raised up, and atmospheric pressure inside an impregnation
the lifting mechanism carefully removes the tissue and embedding oven to hasten the removal of
basket and gently transfers it to the next beaker. the air bubbles
 The machine is mounted on rollers to permit  and clearing agent from the tissue block thereby
turning of platforms and easy access to beakers promoting a more rapid tissue impregnation
and wax bath
 It removes residual bubbles such in the lungs
 Makes use of 12 individual processing steps
with 10 one Liter capacity glass beakers and 2  Particularly recommended for urgent biopsies
thermostatically controlled wax baths with a and for delicate tissues such as lungs, brain,
safety device cut-out switch to protect wax from decalcified bones, eyes, spleen and lymph
over heating nodes
 There is also 2-3 changes of wax needed to o The tissue is then not over-exposed to
remove the clearing agent and to impregnate the heat, brittleness, shrinkage, and
specimen hardening
o This is made possible due to constant
 Time required is reduced from 25% to 75% of
tissue agitation which accelerates and
the normal time required. (Fastest)
improves tissue penetration giving rise
to more consistent results  Composed of flat-bottomed heavy brass
chamber with heavy glass lid with thick rubber
ADVANTAGES OF AUTOMATED PROCESSING valves which produces an airtight seal when the
 It’s very efficient chamber is being used.
 Saves time and energy to operate
 The vacuum chamber is enclosed in a
 Cost effective and user friendly thermostatically controlled water jacket which is
 Can process different tissues same time usually maintained at a temperature 2-4 degree
 The machine does the transfer of tissue from Celsius above the melting point of the wax
one bath to another.
PROCESS
PRECAUTIONS 1. After clearing with xylene
 Presence of any odor of the clearing agent
2. Place tissue in paraffin wax, in vacuum chamber
during final paraffin wax bath indicated that the
and make the oven airtight
paraffin should be changed
 Dehydrating agents should be frequently 3. Exhaust the air slowly by means of vacuum
changed since dehydration is the most critical pump until there is negative pressure of 400 to
stage of tissue processing and inadequate 500nmHg
dehydration is difficult to correct once tissue is in 4. Leave for 15 minutes then slowly readmit air
paraffin until normal atmospheric pressure is reached
 After two complete processing runs of load, the
5. Place the tissue in a fresh wax.
first 100% alcohol bath should be always
discarded and the other moved down so that the 6. Repeat steps 3 and 4
final batch has fresh absolute 100% alcohol o Place the tissue in a fresh wax
 The clearing agent and the other diluted
7. Repeat step 3 and leave for 30-45 minutes
alcohols can be changed at least once a week
 To avoid spillage, fluid and wax containers must 8. Bring to normal atmospheric pressure and
be filled to the appropriate level proceed to embedding
 Wax accumulating on the surface of the beaker
should be removed FACTORS AFFECTING PARAFFIN
 Any spillage should be wiped away IMPREGNATION
 Wax bath thermostat should be maintained at o bone, teeth, brain and eye are difficult to
least 3 degrees above the melting point of the infiltrate (BeeBee) using paraffin (its hard to
wax and should be checked when loading the infiltrate the heart of my Beebee crush)
machine o Require prolonged period of immersion with
VACUUM EMBEDDING paraffin
o REMEMBER: prolonged immersion with paraffin
causes tissue shrinkage

JLLD 18
Histopathology and Laboratory Management

o THUS there are other recommended embedding  More elastic and resilient because of the plastic
medium for these tissues component
o Benzene and Xylene are easily removed from o permitting large dense tissue blocks
the tissues while chloroform and cedarwood oil such as bones, teeth and brain to be cut
requires frequent wax changes. easily with the same result as in double
embedding.
o Adding benzene to cedarwood oil would hasten  Serial sections can be cut with ease without
the process cooling the block, thereby preventing formation
of ice crystals
PRECAUTIONS OBSERVED IN PARAFFIN WAX  It is soluble in common clearing agents and
IMPREGNATION IN GENERAL follows the same time schedule for paraffin
 Prolonged treatment with paraffin causes tissue impregnation and does not tend to crack like
shrinkage and hardening of the tissue, making other paraffin wax substitutes.
cutting difficult
EMBEDDOL
 Overheated paraffin (above 60 degree Celsius)
can cause tissue shrinkage and hardening  Similar to Paraplast
 Paraffin wax must be pure  (less brittle and less compressible than
 Free from dust, water droplets and foreign paraplast)
matter  Can be use as substitute
 Fresh wax should be filtered at 2 degree Celsius  Melting point of 56-58 degree Celsius
higher than its melting point BIOLOID
 Wax that has been trimmed away from the
 Semisynthetic wax recommended for
impregnated tissue may be melted and filtered
embedding eyes
for future use with a coarse filter paper
 For organs with thin wall and circular shaped
ALWAYS FILTER FRESH WAX sections
• If wax is suspected to be contaminated with TISSUE MAT
water, it can be subjected to 100-105 degree
Celsius to remove the water.  Paraffin with rubber
 With same property as Paraplast
• Paraffin wax can only be reused once.
ESTER WAX
PARAFFIN EMBEDDING  Has lower melting point than paraffin (46-48
 Tissue will now be embedded in paraffin wax degree Celsius) but is harder than paraffin
melted at 5-10 degree Celsius above the melting  Main component: Diethylene glycol distearate
point o a very hard and brittle
 Cooling is done:  Added with:
o Refrigeration at -5 degree Celsius o Castor oil
o Immersion in cold water o Ethyl cellulose
o Tissue tek with cold plate o Stearic acid
 Good for sections as thin 2-4 microns
SUBSTITUTES FOR PARAFFIN WAX
DIETHYLENE GLYCOL
1 Paraplast
 Not soluble in water but soluble in 95% ethyl
2 Ester Wax
alcohol
3 Water Soluble Waxes o Tissue can be impregnated without prior
PARAPLAST clearing
o Cellosolve and xylene may be used as
 Paraplast is suitable for tissue infiltration.
clearing agents (depending on the
o (Mixture of highly purified paraffin +
protocol of the hospital or preferences of
synthetic plastic polymers)
the pathologist)
 Melting point: 56-57 degree Celsius
 Clearing agent must be gradually removed
 It is a refined combination of highly purified o Tissue must be placed in a solution
paraffin with plastic polymers.
containing equal amount of clearing
 Paraplast – Paraffin plus Plastic!!! agent and ester wax for 3-6 hrs before
transferring the tissue to pure ester wax

JLLD 19
Histopathology and Laboratory Management

o 3-4 changes of wax should be done to  Tissue block must not have contact with
ensure complete tissue impregnation water or ice
 Sectioning of Ester wax-impregnated tissue  Tissue sections are difficult to float out and
should be done on a heavy duty microtome mount out due to its extreme solubility in
(sledge or sliding type) due to the relative water
hardness of the wax o Add soap in water to reduce
distortion and promote flattening
WATER-SOLUBLE WAXES and floating out of sections
 Composed of polyethylene glycol with  It will not infiltrate tissue however when
melting points of 38-42 or 45-56 degree there is large amounts of fat in the tissue.
Celsius Tissue such as central nervous tissue needs
 Carbowax – polyethylene glycol composed long periods of impregnation. 
of 18 carbons or more which appears solid
at room temperature
EMBEDDING
CARBOWAX
 Also known as Casting or Blocking
 Carbowax is soluble in water hence does  Process by which the impregnated tissue is placed
not require dehydration and clearing of into a precisely arranged position in a mold
tissue containing a medium which is then allowed to solidify
o After the tissue is fixed, it is washed out  The medium used to infiltrate the tissue is usually
to remove excess fixative and the same medium utilized for embedding
precipitates and transferred directly to
melted carbowax  After impregnation, the tissue is placed into a
o Processing time is reduced mold containing the embedding medium and this
 Harmful effects of dehydrating agents and medium is allowed to solidify.
clearing agents are avoided  Paraffin embedded tissues are
 Fats which are usually removed by  arranged at the bottom of the mold and
dehydrating and clearing agents are not immersed in melted paraffin at a temperature
removed between 5 – 10oC above its melting point and;
 Eating too much CARBs can make you FAT! then cooled rapidly
Fat is not removed from the body!!! o Refrigeration at -5oC
o Immersion in cold water
 Tissues are not exposed to too much heat
 This allows hardening of tissues, giving them a
o There is no excessive hardening, firmer consistency and better support, thereby
shrinkage and brittleness of tissue facilitating the cutting of sections.
o Cytologic details are preserved  Orientation - process by which a tissue is
arranged in precise positions in the mold during
 Eating too much CARBs can make you FAT! embedding, on the microtome before cutting,
 No gym workout = No HEAT = No sweat! and on the slide before staining
 No heat = No shrinkage and hardening of  Generally speaking, the surface of the section to
muscle! be cut should be placed parallel to the bottom of
 Suitable for enzyme histochemical studies the mold in which it is oriented.
because enzymes are denatured greatly by
heat SEVERAL TYPES OF BLOCKING OUT MOLDS
 Four changes of carbowax are needed (56 MAY BE USED:
degree Celsius) 1. Leuckhart’s Embedding Mold’
o Once in 70% for 30 mins
2. Compound Embedding Unit
o Once in 95% for 45 mins
o Twice in 100% Carbowax for 1 hr 3. Plastic Embedding Rings and Base mold
each with agitation 4. Disposable Embedding Molds
o Specimen is then embedded in 1. LEUCKHART’S EMBEDDING MOLD’
carbowax and rapidly cooled in
refrigerator  consists of two L–shaped strips of heavy
 Hygroscopic property = easily dissolve in brass or metal arranged in a flat metal plate
water

JLLD 20
Histopathology and Laboratory Management

and which can be moved to adjust the size o Blocks can be filed up after
of the mold to the size of the specimen.  sectioning
 The L- mould are kept on a metal plate. 4. DISPOSABLE EMBEDDING MOLDS
Tissue is placed the surface of the glass and
is oriented  Peel–away disposable thin plastic
 The plate is then filled with molten wax. The embedding molds 
wax is then cooled. L-moulds are then o available in 3 different sizes, are
removed simply needed off one at a time, as
 Easy to procure soon as the wax has solidified,
 Reusable giving perfect even block without
 Blocks produced are even, with parallel trimming.
sides, and with a fairly shaped initial setting  Plastic Ice Trays
of the wax. o used in ordinary refrigerators may
 Adjustable to make different size of blocks be recommended for busy routine
 It is recommended for routine use, although laboratories.
too slow and cumbersome for use in a busy o Each compartment may be utilized
laboratory. for embedding one tissue block,
which may then be removed by
2. COMPOUND EMBEDDING UNIT bending the plastic tray once the
 made up of series of interlocking plates wax has solidified: or by smearing
resting on a flat metal base, forming several the inner mold with glycerin or
compartments. before embedding.
 It has the advantage of embedding more
specimens at a time, thereby reducing the
time needed for blocking  Paper boats 
o are normally utilized for embedding
3. PLASTIC EMBEDDING RINGS AND BASE celloidin blocks but are equally
MOLDS useful for paraffin wax blocks.
 consist of a special stainless steel base o They have the advantage of being
mold fitted with a plastic embedding ring cheap and easy to make. They
which later serves as the block holder during provide easy and accurate
cutting. identification of specimen, thereby
 One model, the so called Tissue Tek, is a avoiding confusion and interchange
machine equipped with a warm plate to of tissue blocks.
manage the impregnated specimen, and a o Rapid embedding of small or large
cold plate at –5oC for rapid solidification of volume of individual specimens is
the block. possible, since paper molds can be
 The base mold is filled up with meltin made to suit any size of tissue.
paraffin.
 The sample is then oriented in the mold DOUBLE EMBEDDING METHOD
 The plastic embedding ring is placed in  Peterfyi's celloidin-paraffin wax technique
position and filled up with wax.  the process in which tissues are first infiltrated
 The tissue is then allowed to cool down on with celloidin and subsequently embedded in
the cold plate of the Tissue tek equipment paraffin mass.
 Upon hardening (5minutes),  This is used to facilitate cutting of large blocks of
o the tissue is taken out together with dense firm tissues like the brain, bone, and teeth
the embedding ring and  also recommended for making small sections of
o is immediately ready for cutting celloidin blocks.
without having to undergo trimming  Gelatin impregnation is rarely used except
thereby saving time and effort. o when dehydration and clearing is to be
 Advantage of Tissue Tek avoided because gelatin is water soluble
o Tissue is firmly attached to the o when tissues are to be subjected to
holder with permanent identification histochemical and enzyme/lipids
o Resectioning is easily done studies.

JLLD 21
Histopathology and Laboratory Management

 Fixatives (such as 10% formalin) should still be  The next day, an equal volume (50 mL) of ether
washed out by running water before tissue is is added and intermittently mixed until an evenly
infiltrated by gelatin consistent solution is obtained
 It is used as an embedding medium for delicate  Dense tissues which are hard to infiltrate (e.g.
specimens and frozen tissue sections bones and brains) and specimen which tend to
o because it prevents fragmentation of collapse easily due to air spaces (e.g. eyes and
tough and friable tissues when frozen lungs) are supported better, thereby avoiding the
sections are cut. crumbling of tissues during sectioning.
 It has a lower melting point and does not cause
overhardening of tissue by heating.  When eye sections are embedded by the
paraffin method, the retina may be detached
 Used along with 1% phenol to prevent growth of
from the harder tissues (e.g. sclera and choroid)
molds
that encircle it.
GELATIN IMPREGNATION  Its rubbery consistency allows tissue blocks that
 After the fixative has been completely washed are either very hard or of varying consistency, to
out, the tissue is be cut without undue distortion.
o placed in 10% Gelatin with 1% Phenol  It permits cutting of tissue sections which are
for 24 hours thicker than paraffin wax, and is therefore
o transferred to 20% Gelatin with 1% recommended for processing of neurological
Phenol for the next 12 hours, and tissues.
o finally to another fresh solution of 20% o Thicker sections contain higher number
Gelatin with 1% Phenol which is then of neurons
allowed to cool in a refrigerator until
impregnation and embedding are
completed.
CELLOIDIN BLOCKS
CELLOIDIN
There are two methods for celloidin impregnation of
 Purified form of nitrocellulose soluble in many tissue
solvents, suitable for the following specimens 1 Wet Celloidin method
o Organs with large hollow cavities (eye, 2 Dry Celloidin method
lungs)
o For hard and dense tissues (bone and WET CELLOIDIN METHOD
teeth)  Recommended for bones, brain sections and
o Delicate tissues (embryos) whole organs.
 It is supplied in  After the usual fixation and dehydration of the
tissue, it is place in equal parts of ether and
o thin (2%), medium alcohol for 12 – 24 hours.
o medium (4%) and o Clearing can be by passed
 The tissue is then placed in thin celloidin (2–4%)
o thick (8%) solutions of cellulose
for 5–7 days,
o dissolved in equal parts of ether and  transferred to medium celloidin (4–6 %) for
alcohol (solvent). another 5–7 days,
 For example, to prepare 8% celloidin  drained off and poured with thick celloidin (8–
12%) until the specimen has become
o Get 8ml celloidin. impregnated, usually between 3–5 days.
o Mix it with 92ml solvent (50 ml ether + o + 7 + 5 = 19 days!!!!! (Slow process)
50 ml alcohol)  The specimen is removed from the celloidin and
Preparation transferred to an embedding medium containing
freshly poured thick celloidin and kept in a tightly
 The fastest way to dissolve celloidin is to soak it covered jar or dessicator in order to evaporate
first in half the final volume of anhydrous ethanol the alcohol – ether solvent.
to soften it (50 mL for each 8 grams celloidin),  The dessicator top is removed for a few second,
with intermittent mixing in a tightly stoppered time and again, to admit fresh air and harden
container. tissue block.

JLLD 22
Histopathology and Laboratory Management

 Evaporation must be gradual to achieve a  Comparing the thin preparations of celloidin and
consistent, uniform degree of hardness LVN, the latter has better tissue penetration or
throughout the block and prevent the formation infiltration
of air bubbles.  LVN is also cheaper than celloidin
 When the ball of the finger leaves no mark on  Celloidin is available as thin (2%) , medium (4%)
the surface of the tissue leaves no mark on the and thick (8%) preparations (lowest viscosity to
surface of the tissue block, evaporation and highest viscosity)
consequently, embedding, is considered to be  The equivalent concentrations for LVN are 5%,
complete. 10% and 20%. 
o Use the thumb, not your fingernails!!!  Note: at same the viscosity, LVN has a higher
 The tissue block is then stored in 70 – 80% concentration. It means it has better tissue
alcohol until ready for cutting. This is done to penetration
avoid dehydration and shrinkage of tissues.  It forms a harder tissue block and makes cutting
 Prolonged contact with air dries out and hardens of thinner sections possible.
celloidin.  Sections tend to crack during staining
DRY CELLOIDIN o The tendency of tissues to crack may be
prevented by adding plasticizers (e.g.
 Preferred for processing of whole eye sections
Oleum Ricini or Castor oil)
 The principle and procedure of this method is  More explosive than celloidin and should
similar to wet celloidin method, except that therefore be handled with care.
70% alcohol is not used for storage before
 When dry, striking or dropping the container will
cutting.
cause the substance to explode.  
 Instead, Gilson’s mixture, made up of equal
o It is usually marketed while wet with
parts of chloroform and cedarwood oil is added
alcohol.
to the celloidin block before hardening.
 The container must be kept tightly covered and
 The cedarwood oil used in the Dry Celloidin
protected from sunlight to avoid evaporation of
Technique helps to soften the layers of eye
alcohol.
preventing the retinal detachment.
o Cedarwood oil is added twice daily for  When no longer needed for future use, the
nitrocellulose should be carefully disposed,
10 days until the mixture is composed of
since the material becomes increasingly
90% percent cedarwood oil.
dangerous as the alcohol continues to
o The purpose of this method is to make
evaporate.
the tissue transparent
o Alcohol is not used in Dry method
because it will dissolve the cedarwood
oil. PLASTIC RESIN EMBEDDING
 Provided superior results for light microscopic
LOW VISCOSITY NITROCELLULOSE
studies
 Another form of celloidin soluble in equal  Thinner sections – high resolution microscopy
concentration of ether and alcohol with a lower such as electron microscope
viscosity, allowing it to be used in higher o Renal biopsies
concentrations and still penetrate tissues rapidly.
EPOXY PLASTICS
 Composed of Epoxy plastics, catalysts and
Take note: accelerators
 Higher viscosity means poor tissue penetration o Araldite (bisphenol A)
or infiltration  slowest, large molecule with
 Higher concentration means good tissue high viscosity
penetration o Glycerol-based (epon)
 Celloidin = higher concentration means higher  faster, smaller size than araldite,
viscosity lower viscosity but cannot be
Conclusion: prepared in pure form
o Cyclohexene dioxide (spurr)
 At lower preparations, both celloidin and LVN  low viscosity and can obstained
have the same viscosity. However, at the same in pure form
level viscosity, LVN has a higher concentration

JLLD 23
Histopathology and Laboratory Management

 Efficiency is based on size of molecules, • Use forceps or brush instead of your fingers to
viscosity and purity pick up sections or wax fragments from blade or
 Hydrophobic – not compatible with most water block face.
soluble stains • The handwheel brake will lock the microtome
 Irritation on skin and airways when the handle is in any position and is used
 Take note: Gloves must be worn while when realigning a block face or adjusting the
processing and processing should be done coarse feed.
inside fume hoods to eliminate toxic vapor and • The knife or blade should be removed from the
avoid irritation to the airways microtome when the instrument is left
 Contains vinylcyclohexene dioxide – unattended or when cleaning the instrument.
carcinogenic - skin cancer • Never place a knife or blade on the bench or in a
 Reduces antigenicity – not recommended for box with the cutting edge facing up.
immunohistochemical studies
MICROTOME KNIVES
 Example: Cytokeratin 18 is overexpressed on
Microtome knives may be generally grouped into:
the surface of cells in colon cancer
1. standard thick metal
ACRYLIC PLASTICS
2. thin disposable blades
 Composed of acrylic or methacrylic acid for light 3. glass knives
microscopy 4. diamond knives
 Less viscous than plastic resins = less time
needed for embedding SET BLADE CLEARANCE ANGLE OPTIMALLY
 Polyglycol methacrylate (GMA) Blade clearance angle is adjustable and must be set for
o Hydrophilic – compatible with water optimum performance.
soluble stains • The clearance angle prevents contact between
 Methyl methacrylate (GMA) the knife facet and the face of the block.
o Preferred for its relative hardness = • The facet angle is the angle between the two
better sectioning facets that form the cutting edge. For routine use
 Prematurely polymerize in the presence of light, knives and disposable blades are made with a
that’s why acrylics should be stored in facet angle of approximately 35°, but this angle
amber(dark) bottle can vary with the blade type and from
manufacturer to manufacturer
• Follow the microtome manufacturer’s guidelines
SECTIONING for the recommended angle setting. For Leica
• Aka microtomy or cutting knife and blade holders a setting of between 1°
• process where tissues are cut into uniformly thin and 5° is recommended.
slices to facilitate microscopic studies
• Instrument: microtome TAKE CARE WHEN “TRIMMING”, “FACING”, OR
“ROUGHING”
LOCATE MICROTOME APPROPRIATELY This stage in microtomy requires great care as tissue of
The location of the microtome in the laboratory is diagnostic importance can easily be lost or the block
important. surface damaged.
• Position the microtome on a stable bench, away • Before you commence trimming always make
from air drafts, doorways and passing staff. certain that all of the clamping mechanisms are
• A height-adjustable bench and ergonomic chair tightened securely
are preferred. • The goal of properly trimming a block is to
conservatively expose the tissue down to a level
UTILIZE SAFETY FEATURES PROPERLY where a representative section can be obtained.
You must be familiar with the safety features of the • Trimming is usually done at thicknesses
microtome you are using and observe some basic rules between 10 and 30 μm.
when cutting sections.
CONSIDER FACTORS AFFECTING SECTION
• Microtome knives and disposable blades are THICKNESS
extremely sharp and can inflict serious injuries Set the microtome at the desired setting but note that
unless appropriate care is taken when working there are a number of factors that determine the actual
with them. section thickness.

JLLD 24
Histopathology and Laboratory Management

• A cohesive section of 4 μm may provide more Proper drying ensures that sections are completely
information than a severely disrupted section of dehydrated, free of heat damage, flat and unlikely to lift
2 μm. during staining.
• The actual thickness of the first couple of • Drain excess water from beneath the section
sections in a ribbon may be thicker than before drying. This is vital if slides are dried flat
indicated because of thermal expansion, when on a hot plate .
cutting a cold block (as seen in sections 1, 2 & 3 • Slides can be stored in racks in an upright
below). - Next picture position, then dried in an oven.
• Other factors such as speed of rotation, • Generally drying temperatures should not
clearance angle setting and the condition of the exceed 65 ˚C.
cutting edge can influence the actual thickness • Excessive heat can cause droplets of water
achieved. underneath a section to boil and this will cause
damage. • Dry sections for between 10 and 30
ENSURE BLOCKS ARE COLD
minutes.
Sectioning is generally improved when the specimen • Some delicate specimens will produce best
and the wax are well matched in hardness. It is for this results when dried at 37 ˚C for a longer time
reason that most paraffin blocks must be cold when (several hours to overnight).
sections are cut. The actual method used to chill the
block is important. CLEAN AND MAINTAIN THE MICROTOME
• Cold wax provides better support for the harder THOROUGHLY
elements in a specimen allowing thinner It is important to remove accumulated tissue debris and
sections to be obtained. wax after use. Regular preventative maintenance is
• Place the blocks on a cold plate or a cold wet important.
surface for a few minutes (such as the surface of 1 Clean the instrument daily.
melting ice). 2 Always remove the knife or blade before
cleaning.
LEARN THE TECHNIQUES FOR CUTTING
3 The knife holder can easily be removed to
CONSISTENT, HIGH-QUALITY, THIN SECTIONS
facilitate access for cleaning.
There is no substitute for experience but there are. some 4 Section waste is best removed with a dry
fundamental steps that will make the task easier. paintbrush.
• Use a section of blade that has not been used 5 Do not clean the outer surfaces with alcohol or
for rough trimming. xylene as they are not resistant to these
• Re-chilling of the block may be required if the solvents and exposure to xylene should be
block face becomes warm or if deeper levels are avoided. Paraffin remover, mild commercial
required. household cleaners or soap and water are
• Generally a slow, uniform cutting stroke recommended.
produces the best results and the least 6 No fluid must enter the inside of the instrument
compression during cleaning.
7 Have the instrument inspected at least once a
FLOAT OUT SECTIONS CAREFULLY year by a qualified service technician.
Floatation should expand the section to its original 8 Follow the lubrication instructions provided in
dimensions and ensure it is completely flat your instruction manual using recommended
• Monitor the temperature carefully. The lubricants.
temperature will need to be 5 - 9 ˚C below the
melting point of the wax.
• Make sure the water is clean and free of bubbles
and section waste (to avoid cross- LEARN TO RECOGNIZE AND CORRECT
contamination). COMMON FAULTS
• Examine each section as it floats on the water Some of the most common faults seen in paraffin
surface as imperfections can be readily seen. sections are:
• Leave the section on the water surface just long
enough for it to flatten. Overexpansion can spoil 1 Wrong micrometer setting
the morphology in susceptible sections 2 First section in ribbon chosen
3 Sectioning at too great a speed
DRY SLIDES ADEQUATELY 4 Poor processing

JLLD 25
Histopathology and Laboratory Management

5 Microtome needs recalibration 3 MAJOR GROUPS/ CLASSIFICATION OF

6 Block trimmed too quickly TISSUE STAINING
7 Block surface not polished by cutting some thin 1. HISTOLOGICAL STAINING
sections after roughing • Tissue constituents are demonstrated by
8 Inappropriate section thickness used when direct interaction with dye/staining solution.
trimming 2. HISTOCHEMICAL STAINING
9 Block brittle (over-processed?) or too cold when • Tissue is studied thru chemical reactions e.
trimmed g. Peri’s Prussian blue for iron/hemoglobin &
10 Damaged knife or blade used PAS for glycogen
11 Poor processing • In Enzyme HISTOCHEMISTRY, the active
12 Hard material such as calcium in block reagent serves as the substrate upon which
13 Debris in unfiltered wax the enzyme act, the final opacity or
14 Buffer salts precipitated in specimens coloration produced from the substrate
15 Poor flotation technique rather than the tissue.
16 Poor fixation and/or processing (insufficient 3. IMMUNOHISTOCHEMICAL STAINING
support) • Allow phenotypic markers to be detected
17 Warm block and demonstrated under the microscope
18 Section too thin using mono or polyclonal antibodies
19 Clearance angle too great
20 Water bath too hot. METHODS OF STAINING
1. DIRECT STAINING
PROBLEMS • Giving color to the sections by using
aqueous or alcoholic dye solutions (e. g.
methylene blue, eosin)
2. INDIRECT STAINING
• Action of dye is intensified by adding
another agent or MORDANT which serves
as a link or a bridge
• MORDANT + DYE = LAKE (tissue –
mordant – dye complex is insoluble)
• ACCENTUATOR – accelerates or hastens
the speed of staining reaction by increasing
staining power and selectivity

INDIRECT STAINING METHOD

1. Progressive Staining
2. Regressive Staining
3. Differentiation (Decolorization)
4. Metachormatic Staining
5. Counterstaining
6. Metallic Impregnation
7. Vital Staining
8. Intravital Staining
9. Supravital Staining

1. PROGRESSIVE STAINING
STAINING
• Stained in a definite sequence, until desired
• Process of applying dyes on the section to see intensity of color is attained.
and study the architectural pattern of the tissue • Once the dye is taken up by the tissue, it is
and physical characteristics of the cells. NOT washed/decolorized.
• Basic dyes – has affinity to acidic part (e. g. • It is somewhat less favored than regressive
NUCLEUS stained with hematoxylin) due to: producing insufficiently intense
• Acidic dyes – has affinity to basic part of the cell staining producing diffused/obscured details.
(e. g. CYTOPLASM stained with eosin) 2. REGRESSIVE STAINING

JLLD 26
Histopathology and Laboratory Management

• The tissue is first OVERSTAINED then • Reagent should not be exposed to sunlight
DECOLORIZED to prevent explosion
3. DIFFERENTIATION (DECOLORIZATION) • Inactivated by NaCI/HCI before discarding
• Selective removal of excess stain, during 7. VITAL STAINING
regressive staining. • The selective staining of living cell
• In general: if the primary stain used is Basic, constituents demonstrating cytoplasmic
the differentiation is caried by an acid structures by phagocytosis of the dye
4. METACHOROMATIC STAINING particle
• Most dyes stain tissue • Nucleus is resistant to vital staining, the
ORTHOCHROMOATICALLY – stain tissues permeability of the dye in nucleus signifies
with same color of the dye CELL DEATH
• Some is METACHROMATIC – stain tissues 8. INTRAVITAL STAINING
different from the color of the dye • Done by injecting the dye into any part of the
• This stain belongs to the Thizine & animal body (intraperitoneal, intravenous)
Triphenylmathane group • Lithium, Carmine, India ink
• All Metachromatic dyes are Cations or 9. SUPRAVITAL STAINING
Basic • Stain living cells immediately after removal from
o Ex. the living body
1. Tolouidine Blue • Thin slices of tissue are placed in staining
2. Cresyl Blue dishes
3. Safranin • Common dyes used are:
4. Basic Fuschin O Neutral red
5. Azure A, B, C O Janus green
6. Methyl Violet/Crystal Violet O Trypan blue
7. Bismarck Brown O Nile Blue
a. Methylene Blue O Thionine
5. COUNTERSTAINING O Tolouidine Blue
• Used to differentiate, to provide contrast and
background
O Cytoplasmic Stains STAINS & STAINING SOLUTIONS
 Eosin Y 1. NATURAL DYES
 Eosin B • Cochineal dyes, logwood dyes, and
 Phloxine B vegetable extracts.
 Picric acid
 Orange B HEMATOXYLIN
 Rose Bengal • Extracted from the core or heartwood of
 Light Green Mexican tree “Hematocylin
 Lissamine Green Campechianum”
O Nuclear Stains • HEMATIN – the active coloring agent
 Neutral Red formed by the oxidation also known as
 Safranin O “Ripening”
 Carmine Hematoxylin • Ripening/Oxidation
 Celestine Blue o May be done by exposing the
 Methylene Blue substance to air and sunlight
 Tolouidine Blue (SLOW)
o May be done by adding oxidizing
agents such as:
6. METALLIC IMPREGNATION  Hydrogen peroxide
• Tissues are demonstrated not by stain but  Mercuric oxide – ripening
by colorless solution of Metallic salts agent of HARRIS
• Producing BLACK DEPOSITS through HEMATOXYLIN
reduction  Potassium permanganate
• Different from stains it doesn’t incorporate  Potassium perborate
with the tissue but it forms ppt. within the  Sodium perborate
tissue (e. g. Gold Chloride, Silver Nitrate)

JLLD 27
Histopathology and Laboratory Management

 Sodium iodate – ripening • Ex. Romanowsky’s stain, Irishman’s stain,

agent of EHRLICH’S Giemsa stains
HEMATOXYLIN
HEMATOXYLIN
COCHINEAL DYES
• Most commonly used for routine histologic
• OLD histologic dye extracted from Female studies
Cochineal bug “Coccus cacti” • Commonly used mordants are alum and iron
• The cochineal dyes when combined with forming lakes
alum (Potassium aluminate sulfate) it will • Aluminum salts are usually colored blue
produce Carmine’s dye is used as a • Ferric salts are colored blue-black.
powerful chromatin/ nuclear dye
• Carmine’s + Picric acid = Picrocarmine
• Carmine’s + Aluminum Chloride = Best A.) ALUMINUM HEMOTXLYIN SOLN’S
Carmine’s used to stain glycogen - Used for progressive, can be used as regressive
ORGEIN but it requires “blueing”.
1. Ehrlich’s Hematoxylin
• Vegetable dye; LICHENS  Hematoxylin is dissolved with
• Colorless, but when treated with NH3 & alcohol
exposed to air it will produce BLUE/VIOLET  Ripened with Na Iodate
color  Best for decalcified specimens
• Used to stain ELASTIC FIBERS and Frozen Sections
2. Harris Hematoxylin
2. SYNTHETIC DYES  Ripened with Mercuric Oxide
• Also known as “COAL TAR DYES” or  Used for routine nuclear staining
“ANILINE DYES”  Routine, alternate for
Definition of terms: Hematoxylin
3. Cole’s Hematoxylin
CHROMOPHORES – are substances that can  Chloral Hydrate is added as
produce visible colors preservative
CHROMOGENS – contains chromophores,  Used for glycogen and
these are different from dye, since the color they Mucopolysaccharides
impart in tissue is not permanent 4. Mayer Hematoxylin
AUXOCHROME – substances that when  Chloral Hydrate is added as
combined with Chromogen may retain its stain preservative
permanently  Used for glycogen and
Mucopolysaccharides
DYE MODIFIERS – substances that affect B.) IRON HEMATOXYLIN SOLN’S
either the color or properties of a dye 1. Weigert’s Hematoxylin
LAKE – the resultant complex of stain-mordant-  With Ferric Chloride
tissue  Demonstrates muscle fibers and
connective tissues
3 CLASSIFICATIONS OF CHROMOPHORES 2. Heidenhain Hematoxylin
A.) ACID DYE  Ripened with Ferric ammonium
• Where the active coloring substance is sulfate
found in the acid component, and combines  Good for demonstration of
with the basic part of the cell cytoplasmic inclusions, muscle
• Ex. Acid fuschin, Picric acid striations, myelin
3. Phosphotungstic Acid Hematoxylin
B.) BASIC DYE  Reddish-brown to purple
• Where the active coloring substance is basic solution
and have affinity to the acidic part of the cell  Used to demonstrate collagen,
• Ex. Methylene Blue nuclei muscle fibers
C.) NEUTRAL DYES
MOUNTANTS
• Formed by combining aqueous solutions of acid • Usually syrupy fluid, preserve slide, and for easy
and Basic dyes, capable of staining cytoplasm handling and storage, prevents distortion during
and nucleus simultaneously and differentially. microscopic exam

JLLD 28
Histopathology and Laboratory Management

• Gives good refractive index

• Slides must be wiped with XYLENE before
mounting
• Once mounted it can incubate @37C for 24 hrs.
• Good mountant should have a refractive index of
1.518

GOOD MOUNTING MEDIUM

1. Avoid distortion of the image, refractive index of
the mountant should be as near as possible to
that of the glass which is 1.518.
2. It should be freely miscible with Xylene &
Toluene.
3. It should not dry quickly
4. It should crack or produce artefactual granularity
on the slide upon drying.
5. It should not dissolve or fade out tissue sections
6. It should not cause shrinkage and distortion of
tissues
7. It should not leach out any stain or affect
staining
8. It should not change color or pH.
9. It should set hard.

2 TYPES OF MOUNTING MEDIUM

• Acqueos Mounting Medium – usually for lipids
because resinous contain xylene which may
dissolve fats
i. Water
ii. Glycerin – refractive index 1.46
iii. Farrant’s Medium – refractive index 1.43
iv. Apathy’s Medium – refractive index 1.52
v. Brun’s Fluid – recommended for mounting
frozen sections from water
• Resinous Mounting Medium – used for prep that
have been dehydrated and cleared
i. Canada Balsam – natural resin extracted
from Canadian tree, Abus Balsamea
refractive index 1.524
ii. DPX – refractive index 1.532
iii. XAM – refractive index 1.52
iv. Clarite – refractive index 1.544

RINGING
Sealing of Margin of the coverslip
 Use to prevent escape of fluid or semi-fluid
samples
 Evaporation of mountant
 To immobilize the coverslip.
 To prevent sticking of the slides upon storage
1. Kronig’s Cement
2. Durofix

JLLD 29