Intro to histopath-Fixation

histopathology

Deals with the preparation for microscopic examination

fresh tissue

Usually examined when there is an immediate need for evaluation

Preserved tissue

Routinely done in the histopathology section

Motion, Mitosis, Phagocytosis, Pinocytosis

Advantages of fresh tissue examination

Autolysis, putrefaction, degeneration

Post mortem changes:

Autolysis/self-digestion

the destruction of tissues by enzymes which are produced by the tissues and eventually liquefy it.It is the first to occur among all post-mortem changes

Putrefaction

The decomposition of organic matter under the influence of microorganisms accompanied by the development of disagreeable odors

Degeneration

A retrogressive pathologic process in cells in which the cytoplasm undergoes deterioration while the nucleus is preserved

Teasing/dissociation

Process wherein selected tissue specimen is immersed in a watch glass containing isotonic salt solution(or ringer's lactate), carefully dissected or separated and examined under the microscope

Squash preparation/crush

Process where small pieces of tissue not more than 1mm in diameter are placed in a microscopic slide and forcibly compressed with another slide

Frozen section

normally utilized when arapid diagnosis of the tissue in question is required, and especially recommended when lipid and nervous tissue elements are to be demonstrated

Smearing

Useful in cytological examination, particularly for cancer diagnosis

Streaking

Rapid and gentle direct or zigzag application to obtain uniform distribution. Too thin or Too thick smears are unsuitable for examination

Spreading

Little more tedious than streaking, but has the advantage in maintaining the INTERcellular relation.

Spreading

Especially recommended for Fresh sputum, bronchial aspirate, and thick mucoid secretion

Pull-apart

The material disperses evenly over the surface of 2 slides. A single uninterrupted motion is applied

Pull-apart

Serous fluid, concentrated sputum, enzymati GIT, and blood smears

impression smear/Touch preparation

Special method where the slide surface is in contact and pressed on the site. Cells may be examined without destroying their actual intercellular relationship and without separating them from their normal surrounding

Fixation

Aka: preservation; the first and most critical step in histotechnology

Preserve the morphologic and chemical integrity of the cell in as life-like manner as possible

Primary aim of fixation

Harden and protect tissue from trauma of further handling as for easy cutting during gross exam

Secondary aim of fixation

Stabilization of proteins

Most important reaction in fixation

Forming crosslinks between proteins

Stabilization of proteins are done by:

Dry out

Leaving a tissue specimen in air causes it to:

Swell

Leaving the tissue in water (hypotonic solution) will cause the cell to:

Shrink

Leaving the tissue in strong salt (Hypertonic solution)will cause the cell to:

Additive

The chemical constituent of the fixative is taken in and becomes part of the tissue through cross-link formation or molecular complex

Formaldehyde, Osmium Tetroxide, Mercuric chloride

Examples of additive fixatives

Non-additive

The fixing agent is not incorporated into the tissue but alters the tissue composition and stabilizes it through water removal

pH: 6.0-8.0

Optimal pH in fixation

Room temp (18-30 degree celcius)

Traditional/Usual temp:

40-42 degree celcius

Tissue processor temperature:

0-4 degree celcius

Electron microscopy and histochemistry temp:

Room temp (18-30 degree celcius)

Temp. for Mast cell for EM:

Rapid at higher temp

Nucleic acid fixation temp:

60 degrees celcius

Formalin heated to ____ leads to rapid dixation of very urgent biopsy specimen

100 degrees celcius

Formalin heated to _____ leads to the fixation of tissues for tuberculosis

1-2mm^2

thickness for tissues in electron microscopy

2cm^2

Thickness for tissues in light microscopy

0.4cm (4mm)

Thin secrion for light microscopy _____ or as prescribed by tissue processor manufacturer

opened;thinly

Large solid tissue, such as uterus should be ______ or sliced ______

10%NBF

Brain is usually suspended whole in ____ for 2-3 weeks

slighly hypertonic

Best results are obtained using _____ solutions (400-450 mOsm)

isotonic/ hypotonic

____ (340mOsm)/____ solutions cause swelling and poor fixation

Sucrose

Added to osmium tetraoxide fixatives for EM

10%

Concentration of Formaldehyde

3%

Concentration of glutaraldehyde

0.25%

concentration of glutaraldehyde for immunoelectron microscopy

24 hours

Most formalin is fixed for _____ hours (washed out)

2-6 hours; one week

Buffered formalin ____ hours up to ____

3 hours

Fixation time for electron microscopy

Shrinkage and hardening

Prolonged fixation may cause ________ and ______ of tissues

not required

If bacteriologic or toxicologic studies, therefore, fixation is _________

1mm/hr

Rate of penetration for fixatives

20:01

fixative to tissue ratio for formalin

5-10x

amount of fixative for osmium tetroxide

Heat, vacuum,agitation, and Microwave

Fixation can be cut down by using __________ (4)

450 watts at 50-55 degree celcius for 1.5-4 mins.

Microwave calibration

2

Size: not more than __ cm^2 in diameter

4

Size: not more than ____mm thick

16-18 hours

Ideal number of hours for fixation:

Histology

science that deals with the microscopic identification of cells and tissues

Histology

microscopic anatomy that focuses mainly is the analysis and identification of cells and tissues

Cytology

science that deals with the study of cells

Pathology

science that deals with the study of characteristics, causes, effects of disease, as observed in the body

Histopathology

study that differentiate normal tissues or cells from abnormal tissues or cells

Pathologist

a medical doctor engaged in the practice of pathology

Pathologist

they are responsible for the examination of tissues and diagnosis or interpretation of results

Medical Lab Scientist / Histologic Technician

they are a talented artist that transform bits of tissue into artistic masterpieces

Medical Lab Scientist / Histologic Technician

they prepare specimens and combines knowledge, creative ability, and sophisticated instruments

prepare specimen

Main Role of Medical Technologist in Histopathology

examine the specimen

Main Role of Pathologist in Histopathology

1. Anatomical Pathology

2. Clinical Pathology

3. Research / Experimental

4. Special Pathology

Divisions of Pathology

Anatomical Pathology

division of pathology that is focused on the morphology or structures of the organs, tissues, and cells

Clinical Pathology

division of pathology that is focused on the biochemistry and results of the laboratory tests obtained in a clinical laboratory

Research / Experimental

division of pathology that is focused on research

Special Pathology

division of pathology that focuses on the diagnosis of diseases; usually combined with other fields of medicine

Surgical Pathology and Autopsy Pathology

Types of Anatomical Pathology

Surgical Pathology

type of anatomical pathology that do biopsy

Autopsy Pathology

type of anatomical pathology that do autopsy

Biopsy

to remove tissue from a living body for diagnostic purposes

Autopsy / Necropsy

an examination of a cadaver to determine or confirm the cause of death

Clinical Pathology

where various standardized procedures are performed in the lab on body fluids for diagnosis of diseases

1. Hematology

2. Microbiology

3. Clinical Chemistry

4. Serology and Immunology

5. Clinical Microscopy

6. Parasitology

Subdivisions of Clinical Pathology

1. Etiology

2. Pathogenesis

3. Morphological Changes

Nature and Cause of Diseases

Etiology

cause of disease

Pathogenesis

development of disease

Morphological Changes

cellular adaptations, cell injuries, and cell necrosis

1. Congenital and Hereditary Diseases

2. Circulatory Diseases

3. Inflammation

4. Degenerative / Retrogressive Diseases

5. Metabolic Disturbances

6. Nutritional Deficiency States

7. Neoplasms

Classification of Diseases

Congenital And Hereditary Diseases

classification of diseases are birth defects that is present immediately after birth of a newborn and observed among newborns

Congenital

it is caused by problems during gestation or pregnancy

Hereditary

it is caused by problems in the genetic makeup of the newborn

Circulatory Disorders

classification of diseases or problems that affect the organs in the circulatory system

Hemorrhage

excessive bleeding

Edema

transportation of fluids from the blood or lymphatic vessels towards tissues

Gangrene

possible death or decay of tissue due to lack of blood supply

Physical

inflammation caused by trauma or injury to a tissue

Infectious

inflammation caused by microorganisms

Degenerative or Retrogressive Diseases

classification of diseases that changes chemically within the cytoplasm

Alzheimer's Diseases

disease when nervous tissues in the brain start to lose a function