IMMUNOPHENOTYPING

1. IMMUNOPHENOTYPING
IMMUNOHISTOCHEMISTRY (IHC)
FLOWCYTOMETRY
IMMUNOPHENOTYPING
PRESENTED BY --- RAJU
MODERATED BY --- DR. CHARU BATRA

2. IMMUNOPHENOTYPING

3. IMMUNOPHENOTYPING
IMMUNOPHENOTYPING IS A TECHNIQUE USED TO
STUDY THE PROTEIN EXPRESSED BY CELLS. THIS
TECHNIQUE IS COMMONLY USED IN BASIC SCIENCE
RESEARCH AND LABORATORY DIAGNOSTIC PURPOSE.
THIS CAN BE DONE ON TISSUE SECTION (FRESH OR
FIXED TISSUE), CELL SUSPENSION, ETC. AN EXAMPLE IS
THE DETECTION OF TUMOR MARKER, SUCH AS IN
THE DIAGNOSIS OF LEUKEMIA.

4. IMMUNOPHENOTYPING ANALYSIS
Antibodies.
Fluorochromes.

5. ANTIBODY
 Highly specific monoclonal antibodies are used
that are produced by cloned antibody secreting
cells.
 Antibodies are based on cluster of differentiation
(CD)- a protocol used for identification and
distinction of cell surface antigens.
 Using CD system we can identify cells by the
presence or absence of particular surface
markers for e.g. CD3+ or CD20- etc.

6. FLUOROCHROMES
 Fluorochromes are substances that can be excited by
certain light source (such as laser) and emit a
fluorescent signal at a single wavelength.
 Fluorescent dyes can directly bind to certain cellular
content, such as DNA and RNA, and allow us to perform
quantitative analysis on individual cells.
 However, in most cases fluorochromes are conjugated
with monoclonal antibodies, which specifically target
cellular antigens/markers.

7. METHODS FOR THE STUDY OF IMMUNOLOGICAL
MARKERS
1.) FLOW CYTOMETRY TO TEST SUSPENSION OF VIABLE
CELLS OF FIXED CELLS .
2.) IMMUNOCYTOCHEMISTRY TO EXAMINE CELLS ON
CYTOSPIN MADE SLIDES OR DIRECTLY ON BLOOD OR
BONE MARROW FILMS.
3.) IHC TO STUDY CELLS IN FROZEN OR PARAFFIN
EMBEDDED SECTIONS FROM BONE MARROW BIOPSY
SPECIMENS OR OTHER HEMOPOIETIC TISSUES.

8. Antibodies conjugated to fluorescent dyes can bind specific
proteins on cell membranes or inside cells. When labeled cells
are passed by a light source, the fluorescent molecules are
excited to a higher energy state. Upon returning to their resting
states, the fluorochromes emit light energy at higher
wavelengths. The use of multiple fluorochromes, each with
similar excitation wavelengths and different emission
wavelengths (or “colors”), allows several cell properties to be
measured simultaneously.
IMMUNOPHENOTYPING ANALYSIS

9. STANDARDIZATION OF IMMUNOPHENOTYPING

10. INTRODUCTION OF IHC
Immunohistochemistry (IHC) combines
histological, immunological and
biochemical techniques for the
identification of specific tissue
components by means of a specific
antigen/antibody reaction tagged with a
visible label. IHC makes it possible to
visualize the distribution and localization
of specific cellular components within a
cell or tissue.

11. BASIC IMMUNOHISTOCHEMISTRY
IHC REQUIRE 3 BASIC ELEMENTS
1) A CELLULAR ANTIGEN OF INTEREST .
2) A PRIMARY ANTIBODY TARGETING THE
ANTIGEN .
3) A DETECTION SYSTEM TO VISUALIZE THE
LOCATION OF THE ANTIGEN – ANTIBODY
COMPLEX .

12. PROCEDURE
1. Wash the frosted slide with Alcohol or Detergent or soap and dry.
2. Coat the slide with Polylysine.
3. Incubate the slide 37c for 24 hrs.
4. Cut 3-5 thick section with microtome.
5. Dewax the slide in incubator 37c for two hrs.
6. Dehydrate with assending grades of alcohol and clean xylene.
7. Wash with tap water.
8. Place the slide in sutiable solution (citrate,EDTA) and in the microwave
antigen retriever for 20 minutes.
9. Cool the slides for 15 minutes.
10. Wash with PBS buffer for 5 minutes x3 changes.

13. 11. Mark a around the section with diamond pencil.
12 Cover the tissue with polydector peroxide blocker for 10 minutes .
13 Wash with PBS buffer for 5 minutes x3 changes.
14 Cover the tissue with Primary Antibody for 1 hrs.
15 Wash the PBS Buffer for 5 minutes each x3 changes.
16 Cover the tissue with secondary Antibody for one hrs,
17 Wash the PBS Buffer for minutes each x3 changes
18 Cover the tissue with DAB chromogen for 10 minutes.
19 Wash with PBS Buffer within 15 minutes.
20 Counter stain with Hemotoxylin.
21 Dehydrate with alcohol and clear in xylen.
22 Mount the slide DPX.

14. IMPORTANT CONSIDERATIONS FOR IHC
Antibody selection
Fixation
Sectioning
Antigen Retrieval
Blocking
Controls
Direct method
Indirect method
Immunoenzyme
Fluorescence
Multiple labeling

15. MONOCLONAL V. POLYCLONAL
Monoclonal
Mouse or rabbit hybridoma
Tends to be ‘cleaner’
Very consistent batch-to-
batch
More likely to get false
negative results
Polyclonal
Many different species
Tends to have more non-
specific reactivity
Can have very different
avidity/affinity batch-to-batch
More likely to have success
in an unknown application

16. FIXATION
 Aldehyde
 10% NBF
 4% formaldehyde with
PBS buffer
 2% formaldehyde with
picric acid and PBS
 The paraformaldehyde
paradox
 Immersion v. transcardial
perfusion
 24-72 hours
 Many others
 Frozen
 LN2
 With or without sucrose
 OCT
 Fix with acetone or
methanol (fix by
coagulation, also
permeabilizes)
 Best for cell membrane
antigens, cytokines

17. SECTIONING
Paraffin
Must heat and process
through xylene and alcohols –
ruins some antigens
Most commonly used
BEST if not stored more than
two weeks – lose antigenicity
after that time
Frozen
Better survival of many
antigens
Poor morphology
Special storage
Cutting difficulty

18. ANTIGEN RETRIEVAL
 HIER
 Use
MW/steamer/pressure
cooker ~ 20 minutes,
slow cool
 Citrate 6.0
 EDTA 9.0
 EDTA 8.0
 Must determine for each
new antibody/antigen
target
 PIER
 Proteinase K
 Trypsin
 Pepsin
 Pronase
 Destroys some epitopes
 Bad for morphology

19. BLOCKING
Background staining
Specific
Polyclonal antibodies – impure antigen used
Inadequate fixation – diffusion of antigen – often
worse in center of large block
Non-specific
Non-immunologic binding – usually uniform
Endogenous peroxidases
Endogenous biotin

20. CONTROLS
Positive control
Best is tissue with known specificity
Negative control
Best is IgG from same species immunized against
non-biologic molecule – e.g. BRDU when no BRDU
is present in tissue
Can also use non-immunized serum from same
species

21. DIRECT METHOD-
PRIMARY ANTIBODY ONLY
Goat anti-actin labeled with
594
Fluorochrome conjugated primary antibody

22. INDIRECT METHOD – PRIMARY AND SECONDARY
ANTIBODIES
Goat anti-actin
Donkey anti-goat
labeled with 488

23. USES OF IHC
1. Used to identify replicating cells (Brdu).
2. Used for identification of carcinomas (Cytokeratins).
3. Used for Hodgkin’s diseases (CD15 & CD30).
4. Used for renal cell carcinoma (CD10).
5. Gastrointestinal stromal tumours (CD117).
6. Prostate cancer (PSA)
7. Yolk sac tumors (Alpha fetoprotien).
8. B cell lymphomas (CD20).
9. T cell lymphomas (CD3).
10.Breast and Gyne. Tumours (ER & PR).

24. FLOW CYTOMETRY

25. COMPUTER
SYSTEM
ELECTRONIC
SYSTEM
OPTICAL
SYSTEM
FLOW
SYSTEM
For cell analysis, the basic components of a flow
cytometer include:-

26. FLOW CYTOMETRY
Definition:
Measuring properties of cell as they flow in a
fluid suspension across an illuminated light path.

27. BASIC MECHANISM
Biological sample
Label it with a fluorescent marker
Cells move in a linear stream through a focused
light source (laser beam)
Fluorescent molecule gets activated and emits
light that is filtered and detected by sensitive light
detectors (usually a photomultiplier tube)
Conversion of analog fluorescent signals to digital
signals

28. FLOW CYTOMETRY
This method allows the quantitative and qualitative analysis
of several properties of cell populations from virtually any
type of fresh unfixed tissue or body fluid.
The properties measured include a particle’s related size,
relative granularity or internal complexity, and relative
fluorescence intensity
Most commonly analyzed materials are:
 blood, bone marrow aspirate and
 lymph node suspensions.

29. PRINCIPLE OF FLOW CYTOMETRY
Flow cytometer is composed of three main
components:
The Flow system (fluidics)
Cells in suspension are brought in single file past
The Optical system (light sensing)
a focused laser which scatter light and emit
fluorescence that is filtered and collected
The Electronic system (signal processing)
emitted light is converted to digitized values that
are stored in a file for analysis

30. ANALYSIS
A measurement for the
diffraction of light in a flat
angle is the forward scatter
(FSC), which depends on
the volume of the cell.
A measurement for the
diffraction of light in a right
angle is the so called side
wards scatter (SSC). It
depends on the granularity
The process of collecting data
from samples using the
flow cytometer is termed
'acquisition‘

31. MULTI ANGLE POLARISED
SCATTER SEPARATION
31
Hydrodynamic focusing

32. ABNORMAL/ ABERRANT ANTIGENIC
EXPRESSION CAN BE GROUPED INTO FOUR
BASIC CATEGORIES:•
•Abnormally increased or decreased levels of antigenic
expression (aberrant expression)
• Gain of antigens not normally expressed in the cell type
• Expression of antigens not synchronized with normal
development and maturation stage of the cell type or lineage
• Homogeneous expression of antigen(s) by a cell population
that normally show more heterogeneous expression

34. LINAGE SPECIFIC MARKERS IN HEMOPOISIS
1) ERYTHROID CD71
2) MYELOID CD 33,13,117
3) B-LYMPHOID CD10,19,20,22
4) T-LYMPHOID CD7,3,5,4,8

35. Comparison of immunophenotypic techniques .
FLOW CYTOMETRY IMMUNOHISTOCHEMISTRY
Shorter turn around time (minutes to hours) Longer turn around time (hours to days)
Less subjective result interpretation Subjective result interpretation
Quantitative results Semi quantitative results
Multiple antibodies/ fluorochromes per test Usually limited to a single antibody per slide
Greater antibody selection Fewer antibodies available
Data/results can be electronically transferred Slides can be shipped by mail or courier service
Need fresh cells or tissue Can use fixed/archived tissue
Limited morphologic correlation Architectural and cytologic correlation
Cannot assess nonviable cells Can assess nonviable “ghost” cells

36. THANK YOU