Ch.

10 Microtechniques
Organization of human body:

Cell →tissue→organ →system →body

Cell:

Single (structural & functional) unit of human body.

Tissue:

Collection of cells. For example, epithelial tissue, connective tissue, nervous tissue, muscular tissue.

Histology:

The branch of anatomy that deals with the study of animal & plant tissues.

History of histology:

It stems from the Greek word “histos” meaning web/tissue & “logia” meaning branch of learning.

Histopathology:

The branch of pathology that deals with the study of diseased tissue e.g. malignancy etc.

Histotechniques/Microtechniques:

The procedures/techniques required to reach the final stained slide of the specimen for microscopic
examination.

Histotechnician:

The lab. technician responsible for histo/microtechniques. (Diploma holder/FSc-MLT)

Histotechnologist:

The lab. technologist responsible for histo/microtechniques. (BS-MLT)

Histopathologist:

The doctor specialized in histo/microtechniques. (M.Phil/FCPS-histopath)

Cytology:

The branch of histopathology that deals with the study of fluid-examination.

Biopsy:

In this test, a small piece of tissue taken out of the body for diagnosis of disease/research.

FNAc: (Fine needle aspiration for cytology)

In this test, a small amount of fluid taken out of the body with the help of a sterile needle/syringe for
diagnosis of disease/research.
EUS-FNAc:

Endoscopic ultrasound guided FNAc under the guidance of Radiologist.

Steps involved in microtechniques

Steps involved in receiving a biopsy specimen are as

1. Documentation/labelling
2. Fixation
3. Grossing
4. Decalcification
5. Tissue processing
 Completion of fixation
 Dehydration
 Clearing
 Impregnation
6. Embedding/blocking
7. Sectioning (3-8µ sized section cutting)
8. Staining
9. Mounting

Fixation
The process, by which the components of the cells/tissues are fixed in their physical/chemical state so
that they can withstand the treatment with many reagents with minimal loss in their structure, is called
fixation.

Routine fixative: 10% formalin

Time: 14-16hrs

Volume: fixative: tissue = 10:1

Purpose of fixation: (changes in unfixed-tissue)

A lot of changes occur if a biopsy specimen is not fixed. So, after the removal of tissue, fixation should be
carried out as soon as possible to

 Prevent autolysis (breakdown of tissue due to the action of enzymes from dead cells)
 Prevent putrefaction (decomposition due to the invasion of tissue by bacteria)
 Prevent the tissue from undergoing osmotic shock (burst/lysis after H2O absorption)
 Prevent distortion of the tissue
 Prevent shrinkage of the tissue

Fixatives:

The chemicals used in fixation, are called fixatives.

Properties of an ideal fixative:

 Prevents autolysis & putrefaction (bacterial decomposition)

 Preserves tissues in their natural state
 Preserves tissue volume
 Fixes all components (protein, carbohydrates, fats)
 Avoids excessive hardness of fixed tissues
 Makes cellular components insoluble to reagents used in tissue processing
 Allows enhanced staining of tissue
 Is non-toxic/non-allergic for the users
 Is cheap/not to be very expensive

Principles of fixatives:

The fixative acts to denature proteins by

 Coagulation process (secondary & tertiary proteins → insoluble gels)

 Additive process (crosslinking of end-groups of amino acids)
 Combination of coagulative & additive processes
 Promote the attachment of dyes to particular cell components by opening up of protein side
groups to which dyes may attach
 Remove bound-H2O to increase tissue refractive index to improve optical differentiation
 Alter refractive index of tissues to improve contrast for viewing without staining

Precautions:

Sample size = 2-3mm³

When tissue is fixed, it is important to keep the sample size small, as increased thickness will retard
fixative penetration.

Volume-range of fixative = 20-25 times the volume of the tissue.

Removal of extra coatings:

The peritoneum/capsule around the tissue should be removed/pierced. The blood & mucus should be
rinsed off with saline.

Cutting material:

The tissue should be cut with a new, sharp razor blade/scalpel, rather than scissors to avoid tissue
damage by squeezing.

Special handling:

Some tissues/organs (e.g. lung, eye etc.) will require special handling to ensure that the fixative reaches
to all internal components.

Care:
Care should be given to ensure that the specimen has one or more cut sides to guarantee good
penetration of the fixative.

Alternative-fixation:

Prolonged fixation may result in the chemical masking of specific protein targets & prevention of Ab-
binding during immunohistochemistry protocols. In such cases, alternative fixation methods may be
used. Therefore, there is no universal fixative, which will serve all requirements.

Types of fixatives
There are 3 main types of fixatives: (PCC)

 Primary fixatives
 Compound fixatives
 Cytological fixatives
1. Primary fixatives:

Consist of a single fixative in solution. These may be in absolute form.

Examples:

 Absolute ethanol
 10% formalin
2. Compound fixatives:

Consist of 2/more fixatives in solution.

Examples:

 Zenker’s fluid
 Helly’s fluid
 Bouin’s fluid
3. Cytological fixative:

Preserve cellular structures/inclusions (e.g. mitochondria). These may be alcohol-based.

Examples:

 Ethanol
 Methanol
 Ether

Classification of fixatives
Criteria:

 Action on proteins (coagulant & non-coagulant fixatives)

 Type of fixative
 Use of fixative
Classification:

There are 3 classifications of fixatives: (TCH)

 Tissue fixatives
 Cytological fixatives
 Histochemical fixatives
1. Tissue fixatives:
a) Buffered formal saline
b) Buffered glutaraldehyde
c) Zenker’s formal saline
d) Bouin’s fluid
2. Cytological fixatives:
a) Ethanol
b) Methanol
c) Ether
3. Histochemical fixatives:
a) Formal saline
b) Cold acetone
c) Absolute alcohol

Common fixatives
1. Routine formalin:

Formalin is sold as 40% W/W soln. of formaldehyde gas in H2O.

Use:

It is used as 10-15% V/V soln. in normal saline/CaCl2 soln. called 10-15% formalin/formal saline.

Advantages:

 Is cheapest.
 Most popular fixative.
 Preserves all elements including fats & keeps phospholipid insoluble in fat solvents.
 Does not precipitate protein but combine with –NH2 group to form an insoluble gel.

Disadvantages:

 Formation of acidic crystals “haematin” in the tissue b/c 10% formalin has acidic pH.
 These crystals interfere in staining.
2. Buffered formalin:

Due to acidic pH of routine formalin (10% formal saline), phosphate buffers are added to make it neutral
fixative so that its disadvantages should be minimized.

Preparation:

To make 10% buffered formal saline, mix

  Pure formalin 10ml
 Sodium dihydrogen phosphate 0.4g
 Disodium hydrogen phosphate 0.65g
 Normal saline up to 100ml

Advantages:

 Tissues can be left in it for longer time period e.g. one year.
 No damage/hardening of tissue.
 Sectioning is easy.
 No formation of haematin crystals.
 A number of staining procedures can be used.

Chemicals used during fixation

1. Ethyl alcohol/ethanol: (C2H5OH)

Use:

Used in 90-100% strength.

Advantages:

 Preserves glycogen.
 Useful for histochemistry (glycogen, uric acid & iron).

Disadvantages:

 Precipitates albumin & globulin but not nucleoproteins.

 Causes shrinkage & hardening of tissue.
 Destroys mitochondria.
 Is a reducing agent and, therefore, cannot be used with chromic acid, chromates & osmium
tetra oxide.
2. Mercuric chloride: (HgCl2)

Use:

Used as 70% saturate/half saturated aqueous soln.

Advantages:

 Valuable for nuclear fixation but rarely used alone.

 Penetrates rapidly, fixes chromatin well & enhances its subsequent staining capability.

Disadvantages:

 precipitates proteins
3. Picric acid:

Use:

Used as 1% saturated aqueous soln.

Advantages:

 Does not harden the tissue.

 Does not affect the staining.
 Preserves glycogen & nearly all other elements.

Disadvantages:

 Penetrates poorly.
 Causes shrinkage.
 Not used alone.
4. Chromic acid:

Use:

Used as a pure chemical/mixture of dichrome & acetic acid (e.g. Zenker’s fluid).

Advantages:

 Preserves most elements.

Disadvantages:

 Is an oxidizing agent & therefore incompatible with formalin & alcohol.

 Weakens nuclear staining by dissolving nucleoproteins.
5. Potassium dichromate:

Use:

Used as 2-3% aqueous soln.

Advantages:

 Is a good cytoplasmic fixative.

Disadvantages:

 Is a bad nuclear fixative b/c it is a weak oxidizing agent & tend to dissolve chromatin.
 Gives chromaffin reaction.
6. Osmium tetra oxide (osmic acid):

Use:

Used as 2% aqueous soln.

Advantages:

 Preserves fats.
 Preserves very fine cell details of organelle e.g. golgi apparatus etc.

Disadvantages:

 Is expensive.
 Is unstable.
  Penetrates badly.
 Rapidly coverts to vapors which are irritating.
 Is a powerful oxidizing agent & therefore incompatible with formalin & alcohol.
 Gives a black precipitate of osmium dioxide with unsaturated fats.
7. B-5 fixative:

Advantage:

 Used for fixation of lymph nodes.

8. Zenker’s fluid:

Advantage:

 Used for bone marrow trephine biopsy

 Also used for Negri bodies.
9. Nitric acid:

Use:

 Recommended for urgent tissue biopsies

 Also used for decalcification of bone marrow trephine biopsy(1-3 days)

Advantages:

 Good for nuclear staining

 Rapidly acting decalcifying agent

Disadvantages:

 Is very reactive so biopsy may be impaired if kept in it for longer time

Factors affecting fixation

1. Size & thickness:

Increase in size & thickness of the tissue inversely affect fixation.

2. Tissues having coatings:

Tissues covered by large amount of mucus/blood fix slowly.

3. Fatty tissues:

Fatty & lipomatous tissues fix slowly.

4. Agitation:

Fixation is accelerated by agitation.

5. Temperature:

Fixation is accelerated by maintaining temp. around 60°C.

6. Time:
Smaller biopsies fix in few hrs. & larger must be cut, open & then put into fixative for 8-12hrs.

7. Volume of fixative:

Fixative must be 10-20 times the vol. of tissue.

8. Cost:

Expensive chemicals must be used in low conc.

9. Penetration of fixative:

Different fixatives have variable penetration e.g.

 Formaldehyde: 0.78mm/h
 Mercuric chloride: 0.5mm/h
 Ethanol: 1.0mm/h
 Potassium dichromate: 1.33mm/h

Fixation containers
Different sized Jars/bottles with screw caps, used for fixation.

1. Large specimens:

These should not be squeezed into small containers. This will result in inadequate fixation & autolysis. If
a specimen is too large, not to fit in container, it should be bought as such to the lab. e.g. Amputated
foot may be wrapped in a rubber sheet.

2. Bulky specimens:

These should be bisected cleanly with a large sharp knife before being placed in fixative e.g. Large
tumors, spleen etc.

3. Solid organs:

Cut slices as big as necessary but not thicker than 5mm.

4. Brain:

For fixing the uncut brain, pass a thick thread under the vessels at the base of the brain. The organ is
gently lowered into a bucket containing the solution & allowed to float with the help of thread.

5. Hollow viscera:

Cavity-containing internal organs such as stomach & intestine should be opened at both ends/cut open
along with their length (stomach should be opened at greater curvature).

6. Small biopsies:

To preserve small biopsies in their original orientation, it is better to place them on a piece of filter
paper & then put into fixative.

Transportation of biopsy specimen to the lab.

The specimen should be taken fresh to the lab./wrapped in moist cotton/normal saline to prevent
autolysis.

Ch. 11 Gross room & specimen handling

Introduction:

In grossing room, Dissection is the 1st step, which is followed by preparation of specimen for
histological/microscopic analysis in histopathology lab.

Safety first & last:

Is very necessary in this process b/c the histopathology dept. is rich in hazards e.g;

 biological hazards(infections)
 chemical hazards
 radiological hazards

Risks:

Reflecting the range of materials used to store, process & analyze tissue;

 Toxic
 Flammable
 Allergenic
 Carcinogenic (cancer-causing)
 Electrical

Other factors that heighten these risks are:

 Sharp cutting implements

 Complex machinery
 Movement of specimen around the laboratory

Safety measures:

 All staff should be aware of safety education to continue professional development

 All staff must wear PPE (personal protective equipment) i.e. gloves, goggles & lab coat
 Every lab. should have accessible & clear SOPs(standard operating procedures)

Specimen reception
A separate room is required for reception which acts as the interface b/w non-lab hospital staff, other
visitors & the pathology lab.

Requirements:

The area must be equipped with appropriate easily cleaned benching, adequate lighting, good
ventilation, safety equipment, disinfectants, absorption granules & protective clothing. In the event of
specimen spillage e.g. body fluids, fixative leakage/other mishap, immediate response by staff in this
area will limit any potential local health risk & prevent risk to other lab personnel (employees). The key
point of this room is to receive samples safely & securely.

Imp. Points:

 Confirm identity
 Assigned a unique lab.
 Clinical history
 Hospital registration no. (MR no.)
 Full name & date of birth
 CNIC
 Contact no. for correspondance

Specimen identification no. (labelling)

Case 1:

For single biopsy specimen (for histopathology)

 123 H/25

Case 2:

For sub-parts of fluid specimen (for cytology)

 123 C/25-a
 123 C/25-b
 123 C/25-c

Imp. Points before dissection

Preserving tissue for other studies:

Prior to fixation, some fresh tissue (unfixed) may be preserved;

 For frozen section→ fresh tissue taken to the lab. for immediate analysis
 For DNA-extraction, cytogenetics & molecular pathology → fresh tissue taken to the lab.
 For personalized therapy → tumor genotyping & characteristics
 For special tests → enzyme assay & mass spectroscopy
 For microbiology assessment → placed the tissue in culture media
 For electron microscopy → placed the tissue in glutaraldehyde fixative

Dissection/Grossing room
Must have

 Electrical & natural lighting

 Ventilation & non-absorbent wipe-clean surfaces
 PPE supply for lab. personnel
 Photography instruments
 Tissue macerators
 Disposable bins
 Comfortable environment permitting undisturbed work by pathologist & support technical staff
 Protective environment for all lab staff from formalin vapors & hazardous fluids
 Ergonomically accessible tools & materials

Macroscopic examination:

Photography & other physical techniques should be done.

Other materials:

Various mechanical/prosthetic implants, foreign bodies, bullets, gallstones & medical devices must be
noted & documented.

Storage capacity:

From grossing/dissection/cut-up/blocking facility must have an appropriate storage area for immediate
clearance of already-examined samples so that the dissection area should be prevented to become
cluttered. Thus the storage area shows sturdy shelving with non-crowded samples.

Table maintenance of dissection room:

Must have availability of

 Wide range of sharp cutting blades along with foreceps, long knives for large specimens
 Ventilated bench
 Clear dissection zone
 Absorbent clothes

Cassettes:

The blocks of tissue taken into standard tissue-cassettes which are made up of plastic (20x20x3mm thick
tissue) & not to be filled by samples permitting fluid circulation for tissue-processing.

Precautions:

 The specimens should be analyzed with only one pot open at any one time
 The request & specimen identity should be checked, ideally by two persons, the dissectors &
their assistant.
 The sample should be described in terms of the nature, shape, size & also any defining
chacarteristics.
 Small biopsies e.g. endoscopic mucosal samples having 3 pieces must be described as, largest
piece of tissue having 3mm diameter.
Margin painting:

The samples have been marked with different colored ink to permit designation of the sidedness of the
samples & easily resection the margins.

Photography:

Photographing the macroscopic specimen, whole or during dissection, is imp. In cases of complex
surgical excision, e.g. Wertheim’s hysterectomy, pneumonectomy & localized samples. It may, later also
be used for analysis/case discussion.

Transportation to tissue processor:

After placing the sample in cassettes, whole batch is transported to automated tissue processor.