Histopathologic and Cytologic Techniques

Introduction in Histopath | Lecture | Lesson 1

INTRODUCTION TO HISTOTECHNIQUES • Processing

• Embedding
➢ What is tissue processing?
• Cutting
• Tissue from the body taken for diagnosis
• Staining
• Must be taken to the histopathology
• Cover slipping
laboratory to produce microscopic slides
that are viewed under the microscope by • Quality Assurance
pathologist. ➢ Specimen and sample are both use to represent
body fluids.
• Focuses in the tissue injury/abnormality
➢ Base on Henry 70% of information to give • Specimen – when the body fluids is not
diagnosis is relied to medical technologists 30% yet processed.
is radiology technologist and other healthcare • Sample – when the body fluid is already
personnel. process
➢ Persons who do the tissue processing and make ➢ Macroscopic is the other for gross.
the glass microscopic slide are histotechnologist. ➢ If the pathologist doesn’t like the cutting of the
➢ Under anatomic pathology – histotechnology histotechnologist, the tissue processing will be
division repeat in the cutting part.
➢ Tissue processing is step by step procedure ROLES OF THE HISTOTECHNOLOGIST
TISSUE PROCESSING ➢ Adhere to policies and procedures
1. Fixation – important step; it maintains the tissue ➢ Report problems (reagent supply, equipment
integrity malfunction, poor sectioning by residents)
2. Dehydration immediately to pathologist
3. Clearing ➢ Perform PROPER histopathologic techniques
4. Infiltration/Impregnation ➢ Autotechnicon – an automated equipment that do
5. Embedding step by step tissue processing; machine
6. Trimming ➢ Submit best quality slides to pathologist with
7. Cutting/Sectioning attention to PROPER numbering, labeling,
8. Staining sequence of slides corresponding to specimen
9. Mounting requests and slides free of excess adhesive dirt or
10. Labeling paraffin.
➢ Assist the pathologist in maintaining quality
➢ Decalcification practices in the histolab.
• Is carried out after fixation ➢ From stone age to molecular diagnosis, urinalysis
(urine) is the oldest and considered to be the
• It is only done if the tissue is hard
foundation of laboratory test.
• It is not part of the general steps
➢ Basic Points HISTOPATHOLOGY
• Specimen quality – checked by medtech
➢ Study of the abnormal, diseased tissues
& pathologists/surgeons
➢ It is performed by examining a thin tissue section
• Fixative quality and quantity
under light microscope.
• Fixation times – period of adding fixatives
➢ Usually diagnose malignancy/malignant cysts
• Grossing and sectioning – performed by
➢ A.k.a microscopic anatomy
the pathologist; tissue slide preparation
➢ Benign – non-cancerous/ non-malignant
condition
➢ Malignant – cancerous type
➢ Histopathologic Examination of Tissue Biopsy
• is considered as the gold standard
method or stage for detecting early stage
of cancer.
➢ Oncologists – specialize field in studying of
cancer
➢ Tumor markers 2.2 Excisional Biopsy – the removal of entire
• These are substances that are increase organ 2-3mm (eg. Removal of appendix)
in the blood or in the tissues when there
is a cancer
• Use also to diagnose cancer
• Advanced on early or late stage of cancer

HISTOTECHNIQUES

➢ Art and science performed by the medical

technologist/histotechnologist to produce a
tissue section of good quality that will enable the
pathologist to diagnose the presence or absence
of a disease or malignancy. 2.3 Needle Biopsy – this involves the use of
special type of needle by which tissues can
Specimens Submitted for Histopathological be removed/taken from the organ without
Examination making an incision. Fine Needle Aspiration
Tube (FNAB)
1. Autopsy – tissues derived from a dead body;
2.4 Aspirational Biopsy – this involves the use
cadaver; corpse
of ordinary syringe and needle to aspirate
• is a systematic dissection of a dead body
from a cyst to find out the nature of its
to determine the actual cause of death
content.
2.5 Smear Biopsy/Exfoliative Biopsy – this
involves the scrapping of cells from lining
epithelium
➢ In handling pathologic specimens for
histotechnique processing, the following
criteria are observed:
1. The specimen cannot be accepted in the
pathology laboratory unless formal request has
been properly accomplished.
2. Biopsy/Surgical Specimens – derived from
2. Immediately upon receipt of the specimen, a
living source; collected and transported to
laboratory number is assigned and recorded in the
histopathology lab
logbook with proper checking to avoid confusion
2.1 Incisional Biopsy – the removal of a part of a
and possible interchanges.
whole organ (eg. Small punch of biopsy
collected from a suspected tumor in an The following are to be recorded in the logbook:
organ)
➢ Patient’s whole name
➢ Birthday
➢ Age and sex
➢ Address
➢ Civil status
➢ Tentative diagnosis (if there’s any)
➢ Examination desired FACTORS INVOLVED FIXATION
➢ Name of the requesting Physician
➢ Hydrogen Ion Concentration (pH) – the acidity of
3. Any specimens which are not in good condition
basicity of a cell could be damaged or preserved?
must reported to the pathologist as soon as
7.35 – 7.45 (neutral)
possible.
➢ Temperature
4. The pathologist must be notified when a
➢ Thickness of section
specimen is received for the proper description
➢ Osmolality
and other procedures before fixation.
➢ Concentration
5. Careful processing must be done with great easy
➢ Duration of fixation
by the technologist.
6. All infectious specimens, such as tuberculosis PRACTICAL CONSIDERATION OF FIXATION
specimens, should be placed in an antiseptic
solution such as formalin and Lysol. ➢ Speed
➢ Penetration (other tissue)
Steps in Routine Histotechnique/Tissue ➢ Volume (of fixative to be used)
Processing ➢ Duration of fixation
1. Numbering – is one of the first important steps in EFFECTS OF FIXATIVES
all histopathologic techniques. Its purpose is to
identify properly all specimens without writing the ➢ Harden soft and friable tissue
name of the patient on the accompanying tag. ➢ Makes cell resistant to damage and distortion
- Extra care should be practiced when assigning ➢ Inhibit bacterial decomposition
specimen number since it may lead to mislabeled ➢ Increase optical differentiation of cells and tissue
specimens causing important patient components
assessment and treatment confusion. • Good fixation, gives good outcome
- Histopath number is a unique code; not same ➢ Acts as mordant or accentuator
with other patient. • Mordant means an agent that will bridge
Eg. Succesion Number stains and tissues
Section | Number | Year ➢ Reduce risk of infection
(Autopsy) A – 001 – 24 • Fixatives destroys bacteria
(Biopsy) B – 001 – 24
(Cytology) C – 001 – 24

I-FIXATION

➢ First and the most critical steps in

histotechnology which involves fixing or
preserving fresh tissue for examination.
➢ The primary aim of fixation is to preserve the
morphologic and chemical integrity of the cell.
➢ The secondary goal of fixation is to harden and
protect the tissue from trauma or further handling. CHARACTERISTICS OF A GOOD FIXATIVE
➢ Tissues should be fixed.
➢ Cheap
➢ Stable (has no chemical reactions that could
affect/interfere unnecessary reactions)
➢ Safe to handle
➢ Must kill the cell quickly producing minimum
distortion of cell constituents
➢ Must inhibit bacterial decomposition and
* Histowave
autolysis
➢ Must permit rapid and even penetration of tissue ➢ Cytoplasmic Fixative
➢ Must produce minimum shrinkage of tissue • Regaud’s fluid
➢ Must be isotonic (equal intercellular & • Orth’s fluid
extracellular level of cell) ➢ Histochemical Fixative
➢ Must make cellular components insoluble to • 10% Formal Saline
hypotonic solution • Absolute Alcohol
➢ Must permit the subsequent application of • Acetone
staining procedure • Newcomer’s fluid
➢ Lipid Fixation – use to affix fats; utilizes aldehyde
• Baker’s Formol Calcium – use to preserve
phospholipids
➢ Carbohydrate Fixation
• Alcohol Fixatives are used; generally
used in fixing glycogen
• Glycogen – storage form of glycose in the
liver and muscle
➢ Protein Fixation
• Neutral Buffered Formalin
• Formaldehyde
FIXATIVE ACCORDING TO ACTION AND ➢ Glycogen Fixation
COMPOSITION • Alcohol based fixatives
A. Formaldehyde
I. According to Composition • One of the most widely used fixative for
A. Simple Fixative
tissue fixation, usually buffered to a pH of
1. Aldehydes 7 with the addition of phosphate buffer.
2. Metallic fixatives Advantages:
3. Picric acid
• Cheap, readily available, easy to prepare
4. Acetic acid
and stable
5. Acetone
• Compatible with many stains
6. Alcohol
• Doesn’t overhanded tissue
7. Osmium tetroxide
• Penetrates tissue well
8. Heat
B. Compound Fixative – composed of two • Preserves fats and mucin
or more components • Preserves glycogen
II. According to Action • Preserves but doesn’t precipitate protein
➢ Microanatomical Fixative • Allows natural tissue colors to retained
• 10% Formalin after fixation
• 10% Neutral Buffered Formalin Disadvantages:
• Heidenhain’s Susa • Fumes are irritating to the nose and eyes
• Formol Sublimate • Irritating to skin
• Zenker’s Solution • May produce considerable shrinkage of
• Zenker’s Formol (Helly’s Solution) tissue
• Boiun’s solution • Soft fixative and doesn’t harden some
cytoplasmic structure
• Brasil’s solution
➢ Cytological Fixative
• Nuclear Fixative
• Carnoy’s fluid
• Bouin’s fluid