Teknik Dasar Riset Biomedis

Pembuatan Sediaan Histologi

Teknik Pewarnaan ImmunoHistoChemistry
Flowcytometry
DNA Isolation
Chromosome Analysis
Polymerase Chain Reaction
Electrophoresis
 Introduction

• There are 3 main techniques which are used in

preparing microscopical sections from tissues:

• The paraffin technique (It is the most common

method)

• The celloidin technique (It is the most perfect method)

• The freezing technique (It is the most rapid method)

…Introduction
• Tissues from the body taken for diagnosis of disease processes must
be processed in the histology laboratory to produce microscopic
slides that are viewed under the microscope by pathologists.
• The techniques for processing the tissues, whether
• Biopsies,
• Larger specimens removed at surgery, Or
• Tissues from autopsy are described below.
• The persons who do the tissue processing and make the glass
microscopic slides are HISTOTECHNOLOGISTS.
 Surgical Specimen

• Clinical Details
• Adequate specimen
• Proper Fixative
• 10% buffered Formalin
 Gross Examination:

Description:
• Specimen weight &
measurement (approx)
• Consistency
• Photo *
• Cut section
 Taking Samples:

Edge of lesions.
• Wall of cysts.
• Include normal areas.
• Avoid necrotic area.
• Whole specimen if
small.
• Direction, mark
 Inking the Margins*
• To mark surgical margin.
• Spread of lesion
• Malignancy
• Adequacy of removal
• Different colors to
identify margins
 Fixation:

• Specimen bits are placed

in porous cassettes
• Not more than 5mm
thick
• In 10% formalin
• 1mm/hour fixation
• ~ 6 hour
Fixation:
• After fixation is
Replacing aqueous
formalin with alcohol
in gradual sequence
(70, 95, 100%) to
make way for paraffin.
Clearing:

• Removal of alcohol
with “xylene” that will
be miscible with the
embedding medium
(paraffin)
• Impregnating with
paraffin.
Embedding:
• Paraffin block with
embedded tissue
• consistency to cut
• Paraffin blocks taken for
sectioning
 Sectioning:
• Microtome
• 3-10 microns
• Ribbon of sections
• taken on hot water bath
Picking up sections:

• Floating sections
onto slides
• Common “float”
artefact
Microscope slide preparation:
• Taking the section onto
slide
• Flat, no air bubbles, no
stretch or breaks.
 Automated Staining:

• Routine stain H&E

• Hematoxylin (basic)
• Eosin (acidic)
• Nucleus is acidic and
cytoplasm is relatively basic
• Special stains
Coverslipping:
• Clearing - xylene
• Thin glass coverslips to
protect the section
• Using mounting media
(Eg. DPX, Resins, Canada
balsam etc.)
Reporting:
• Additional sections
• Deeper / Thinner sect.
• Special Stains/tech.
• Reference..
• Discussions with Clin.
• Diagnosis
• Report Typing
• Despatch.
• (>3-5 days)
Principle
• The principle of immunohistochemistry is to localize

antigens in tissue sections by the use of labeled

antibodies as specific reagents through antigen-antibody

interactions that are visualized by a marker such as

fluorescent dye, enzyme, radioactive element or

colloidal gold.
Teknik imunohistokimia
Ada dua komponen utama
• Antibodi primer
• Sistem deteksi  identifikasi hasil reaksi antigen-
antibodi
- sistem pewarnaan (chromogen)
- komponen jembatan/penghubung (antibodi sekunder)
Sistem Deteksi
• 2 komponen: antibodi sekunder / bridging antibody dan
color development system (sistem pewarna)
• Antibodi sekunder menghubungkan antibodi primer
dengan sistem pewarna  antibodi sekunder (goat
antimouse IgG) merupakan antibodi terhadap
imunoglobulin dari antibodi primer (mouse IgG)
• ABC + DAB/AEC

• Substrat + H2O2 + chromogen (DAB/AEC)

 Method
• Direct Method

• Indirect Method

• PAP Method
 Direct Method

Labeled Antibody

Tissue Antigen
 Two-Step Indirect Method

Secondary Antibody

Primary Antibody

Tissue Antigen
PAP Method
(peroxidase anti-peroxidase method)
 General
Immunohistochemistry
Protocol
 Part 1 Tissue preparation

1. Fixation
Fresh unfixed, fixed, or formalin fixation and
paraffin embedding
2. Sectioning

3. Whole Mount Preparation

 pretreatment
Part 2
1. Antigen retrieval

Proteolytic enzyme method and Heat-induced method

2. Inhibition of endogenous tissue components

3% H2O2, 0.01% avidin

3. Blocking of nonspecific sites

10% normal serum

Part 3 staining

• Make a selection based on the type of specimen, the

primary antibody, the degree of sensitivity and the

processing time required.

Controls
• Positive Control

It is to test for a protocol or procedure used.

It will be ideal to use the tissue of known positive as a

control.

• Negative Control

It is to test for the specificity of the antibody involved.

kekeliruan interpretasi
• Misinterpretasi:
• negatif palsu: hilangnya antigenisitas (prosesing),
hilangnya aktivitas antibodi primer
• positif palsu: non-spesific staining
• Hasil palsu karena terjadi sesuatu pada:
• Jaringan
• Antibodi primer
• Antibodi sekunder
• Sistem deteksi
• Perlu kontrol eksternal dan internal
 kekeliruan interpretasi
• Hasil palsu karena terjadi sesuatu pada:
• Jaringan: antibodi terlalu pekat, antigen masking  HIAR
• Antibodi primer:
- bila kurang spesifik  non-specific binding  false positive
(terutama antibodi poliklonal)
- antibodi rusak
• Antibodi sekunder:
- = antibodi primer
- tidak cocok  false negative
• Sistem deteksi: endogenous peroxidase activity
Hasil Positif Palsu
• Reaksi silang dengan antigen yang lain
• Pengikatan non-spesifik antibodi kepada jaringan
• Adanya peroksidase endogen dalam elemen selular
• Jaringan normal terperangkap di dalam jaringan
tumor (jar.otot dalam tumor jaringan lunak  rhabdo-
myosarkoma; jaringan tiroid dalam limfoma maligna 
dikira karsinoma tiroid; dll.)
• Lepasnya protein sitoplasma sel normal karena invasi
tumor  permeasi ke dalam sel interstisial atau
fagositosis  positif palsu
Hasil Negatif Palsu
• Antibodi tidak cocok, rusak, atau konsentrasi tidak
tepat
• Antigen hilang/habis karena otolisis atau difusi,
misalnya bila jaringan terlalu lama dalam formalin
 lebih baik dalam blok
• Keberadaan/densitas antigen di bawah level
kemampuan deteksi oleh reagen atau teknik yang
digunakan, ini karena produksi yang rendah atau
hilang
FLOWCYTOMETRI
 What is flow cytometry?

Flow cytometry is a method of measuring

multiple physical and chemical characteristics of
particles by optical means.

• Peripheral blood, Bone marrow cells

• Bacteria
• Yeast
 Applications of Flow Cytometry.

• Cell size.
• Cytoplasmic granularity.
* Cell surface antigens (phenotyping).
* Apoptosis.
* Intracellular cytokine production (IFN; IL-12).
• Intracellular signalling.
• Gene reporter (GFP).
• Cell cycle, DNA content, composition, synthesis.
• Bound and free calcium.
• Cell proliferation (BRDU and CFSE)
*Cell sorting, single cell cloning (clonecyt)
White Blood Cells, Platelets
( s t a i n e d p u r p l e ) , a T-
Lym phoc yt e w hi t e cell
(stained green), and a
Monocyte white cell (stained
gold) as seen through a
s c a n n i n g
electron microscope.
Flow Cytometry
Action
** Remember you will need to record the number of each cell in
your sample.
• Neutrophils
• Lymphocytes
• Monocytes FLUORESCENT MARKERS !
• Eosinophils
• Basophils
• NK1.1
• Fluorescence Activation Process (or Immunofluorescence

Antibodies recognize specific Antibodies are artificially

molecules in the surface of conjugated to
some cells fluorochromes

FITC
 Phenotyping, Size and granularity detection:
Sample requirements:

A suspension of single cells or other particles

in a suitable buffer, usually PBS.

Typical density : 105 - 107 cells / mL

+
Incubate Acquire
 A cytometer consists of:

Electronics

Fluidics
Light detectors
FL3 PerCP, Cy5

FL2 PE
Computer

FL1 FITC

SSC

Laser
Light FSC

488nm
• Each cell generates a quanta of fluorescence

• Photomultiplier Tubes (PMT’s)

PE FL FITC FL 488nm Sct

Discriminating Filters

Dichroic Lenses Confocal Lens Forward Light Scattering

Detector
Negative cells are also detected

PE FL FITC FL 488nm Sct

Forward Light Scatter

Dichroic Lenses
Confocal Lens
 Optical Bench Schematic
FL3
Sensor FL4
FL2 620BP Sensor
Sensor
675BP
575BP
FL1
Sensor
525BP

SS
Sensor

645DL
Fluorescence
Pickup Lens
600DL

550DL
Laser
Beam
488BK

488DL

Flow
Cell
FS
Sensor
Spectra of Fluorochromes Dyes Used on Research Cytometers - 2
Excitation - 488nm [Argon] / 535nm [HeCad]