IMMUNOHISTOCHEMISTRY AND IMMUNOPATHOLOGY PRACTICE IN PULMONARY PATHOLOGY Dr. Kemal BAKIR Gaziantep...

11TTS 10th Annual Congress April 25th-TTS 10th Annual Congress April 25th-

29th 2007 - ANTALYA29th 2007 - ANTALYA

IMMUNOHISTOCHEMISTRY AND IMMUNOPATHOLOGY PRACTICE IN PULMONARY

PATHOLOGY

Dr. Kemal BAKIR

Gaziantep University, Medical Faculty, Department of Pathology

22

PRESENTATION

History

Aim

Methods

IHC in Pulmonary Pathology

Pitfalls in IHC

Conclusion

33

HISTORY:

Diagnostic immunochemistry was first introduced in 1940s by Coons et al who defined bacterial antigens and fluorescein-binded antibodies in infected tissues.

Being used for the last 30 years, improvement of immunohistochemistry (IHC) occured at the end of 1980s and the beginning of 1990s.

Although a few antibodies were included in the panel at the beginning, thousands of antibodies are now used in routine practice.

44

Immunopathology:

•immunohistochemistry,

•immunofluorescein,

•in situ hybridisation.

55

Immunopathology can be applied to

•Tissue sections,

•Aspiration materials,

•Fluids such as bronchoalveolar lavage

66

77

Tissue antigen

Rabbit immünoglobulin GSheep anti-rabbit IgG

Label (fluorescein, ferritin, enzyme)

88

Direct method

99

Two-step indirect method

1010 Three-step indirect method

1111

Soluble Enzyme Immune Complex Technıques

1212

1313

1414

1515

1616

In situ hybridisation (ISH) is a system depending on a labelled riboprobe, that is complementary to a unique nucleic acid sequence (which is tried to be evaluated or to be shown) in target cell or organisms

Probes which are used in (ISH) may also be labelled with digoxigenin. The probe can be detected using enzyme-labeled anti-digoxigenin antibody.

This technique also allows precise localization and can be quantitatively assessed.

1717

History

Aim

Methods

IHC in Pulmonary Pathology

Pitfalls in IHC

Conclusion

1818

When IHC used?

• confirm the diagnosis with HE • contribute to etiopathogenesis • establish various cell types and areas of

accumulations in inflammatory processes • manage treatments and to investigate new

treatment modalities • evaluate prognostic parameters• get information about mechanisms of rejection and

damage in transplantations

1919

History

Aim

Methods

IHC in Pulmonary Pathology

Pitfalls in IHC

Conclusion

2020

There are many steps in IHC :

Routin Tissue Processing Practicing of IHC Interpretation of IHC

2121

Routin Tissue Processing

•Tissue fixation ( Type and duration of fixation) •Tissue processing•Deparaffinisation

2222

Practicing of IHC

•Antigen retrieval •Staining method and protocol•Validity of solutions used in the staining procedure •Controls

2323

Interpretation of IHC

•Working of the technician and pathologist in harmony •To choose the antibody panel•Sensitivity of the antibody panel •Clone of the antibody•Right interpretation, scoring and evaluation of the positivities.

2424

2525

2626

2727

Detection Systems:

PAP

APAAP

(Strept)avıdın-bıotın

Polymer-based Technology

Staınıng Of Multıple Tıssue Markers At The Same Tıme

2828

Although IHC is generally used with avidin-biotin based detection systems, polymer-based detection systems are now being used because of false results due to binding of biotin with cells especially in kidneys and liver.

2929

It’s supposed that tyramin-based amplification systems will be used more efficiently in the future

3030

Immunohistochemical reactivity is observed in cells and in stroma.

The reactivity in cells may be membranous, cytoplasmic and/or nuclear .

The reactivity is assesed considering either the staining extent or intensity (or a combination of both), and the results are evaluated in a wide spectrum, with ‘positive’ at one end, and ‘negative’ at the other (e.g. Weak, intermediate, strong reactivity; or +,++,+++…)

3131

membranöz

3232

Nükleer ve sitoplazmik

3333

History

Aim

Methods

IHC in Pulmonary Pathology

Pitfalls in IHC

Conclusion

3434

When pulmonary pathology is considered; IHC may be helpful in:

Diffuse parenchymal pulmonary disorders,

Diseases such as COPD and asthma,

To assess the number and types of inflammatory cells

Infective diseases

Benign and malignant pulmonary neoplasia

Mesothelial lesions

Experimental studies cocerning pathogenesis and treatment modalities.

3535

In Asthmatic Patients;

Chronic T-cell mediated immunologic reaction on the bronchial wall of asthmatic patients was investigated immunohistochemically.

T-cells (CD2, CD5, CD7 and CD8 monoclonal antibodies),

B cells ( CD19, CD20 monoclonal antibodies),

Monocytes and macrophages (CD 68),

Eosinophils (EG1) and

Activated eosinophils (EG2) were tried to be shown and assessed quantitatively.

3636

In addition, CD4:CD8 ratio was determined by a double staining immunopathology procedure to assess T-cell subtypes.

3737

The number and ratio of T-cells, eosinophils and macrophages can be established in biopsies from proximal airways and bronchioloalveolar lavage (BAL) from distal airways.

In sudden deaths due to asthma, T-cell lymphocytosis is increased in BAL.

This increase was not observed in asthmatic patients and it was relatively preserved as normal. CD4:CD8 ratio is reversed with an increase in CD8+ T-cells.

3838

Eosinophilia and immunohistochemical nitric oxide positivity in

sputum,

lavage and

BALspecimens

are helpful in differential diagnosis of eosinophilic bronchitis and asthma which have similar clinical presentations.

3939

CD8+ T-cells were investigated in patients with chronic obstructive pulmonary disease (COPD). When compared with non-smokers, an increase in CD8 was observed in smokers.

The role of smoking in pathogenesis of COPD was demonstrated by this way.

4040

Immunopathology, especially IHC also helps in the diagnosis and follow-up of diffuse parenchymal pulmonary diseases

4141

IHC is also used in experimental studies which investigate pulmonary pathologies in humans and try to develop new therapeutic approaches.

One of them is to determine the accumulating cell type and localisation in pulmonary hypersensitivity.

•Dendritic cells tend to accumulate arround bronchioles

•Macrophages prefer arteries

•CD4+ T cells tend to accumulate at the same locations with dendritic cells.

4242

Lung cancer is the first leading cause of death due to cancer worldwide.

Non-small cell lung carcinoma (NSCLC) accounts for more than 80% of all lung carcinomas.

For this reason, it’s essential to make the differential diagnosis of these tumors and predicting the prognosis.

4343

β-catenin is used in differential diagnosis of adenocarcinomas.

Positivity of c-kit is helpful in the differential diagnosis of adenocarcinomas, on the other hand it’s also related with prognosis.

4444Β-katenin

4545

IHC is also used to determine prognostic factors in NSCLC.

These molecular prognostic factors are classified as: 1. Growth factors and receptors, oncogenes2. Programmed cell death3. Regulation of cell cyclus4. Angiogenesis and tumor progression

4646

4747

4848RNP

4949

Another field of IHC is the differential diagnosis of mesothelioma from adenocarcinoma. For this purpose;

calretinin, cytokeratin5/6, podoplanin, WT1 and also

MOC-31, Ber-EP4, B72.3, CEA, BG-8 and TTF-1 may be used.

5050

.

MOC-31,

Ber-EP4,

estrogen receptors and

calretinin

are known to be useful markers in differantial diagnosis of peritoneal mesothelioma and serous carcinoma.

5151

MOC-31,

Ber-EP4,

CEA,

BG-8 and

p63

are all expressed in squamous cell carcinomas and may be used in differantial diagnosis of epitheloid mesothelioma and squamous cell carcinomas.

5252

Mesothelial markers D2-40 and podoplanin, which are being used recently, show positive staining in 90% of epithelioid mesotheliomas.

5353

D2-40 AC-Adeno D2-40 Mesotelioma

5454

Immuncytochemistry (ICC) is a procedure applied to effusion cytology samples and especially to

•Smears,

•Cytospin

•Thinprep preparations.

Since there are problems in application and interpretation of cytologic samples, cell blocks of these materials are preferred for ICC.

5555

5656

Most appropriate samples for ICC are provided by cell blocks.

Also in pulmonary pathology,

Cell blocks are prepared from - sputums,

- bronchial washings and - pleural fluids

so that sections showing cells together which are appropriate for ICC can be obtained.

5757

History

Purpose

Methods

IHC in Pulmonary Pathology

Pitfalls in IHC

Conclusion

5858

Technical pitfalls•Fixation•Tissue processing •Antigen retrieval•Detection systems

Professional pitfalls •Choice of panel•Sensitivity of panel•Selection of type of antibody•Selection clone of the antibody•Interpretation of the results•Interpretation of the literature

5959

Tissue Fixation:

Rapid fixation (drying of the tissue or solution without fixative)

Type of fixation (formalin, B-5, Bouin, glycol-based fixative)

Duration of fixation (a few hours for small tissues)

6060

Tissue processing:

Temperature of the paraffin

Thickness of the sections

6161

Detection Systems:

Avidin-biotin

Dekstran-polymer

6262

Antigen Retrieval:

Enzyme

Temperature

Tamponade

6363

Choosing a panel:

DiagnosisDiagnosis Panel usedPanel used Panel preferredPanel preferredCarcinomaCarcinoma PancytokeratinPancytokeratin PancytokeratinPancytokeratinLymphomaLymphoma CD45CD45 CD45 + CD43CD45 + CD43MelanomaMelanoma S-100S-100 S-100 and Melan AS-100 and Melan ASarcomaSarcoma VimentinVimentin Kollajen type IV and VimentinKollajen type IV and Vimentin

Algorhythm is important in choosing a panel.

6464

Selection of clone of antibody:

Each clone has different sensitivity

In studies using different types of clones, results of IHC staining showed marked variations.

6565

Variations in interpretation:

Technical findings and the literature should be evaluated together.

Shoud be evaluated together with the controls.

6666

History

Purpose

Methods

IHC in Pulmonary Pathology

Pitfalls in IHC

Conclusion

6767

HE for tissue samples

Light microscopic examination of Papanicolau, Giemsa and HE-stained preparations for cytopathology are still golden standarts

Ancillary methods such as IHC and ICC are obviously helpful in

confirming the diagnosis,

contribute to ethiopathogenesis,

selection of treatment modalities and follow up.

6868

6969

Working in harmony with the technician

Right fixative

Right IHC detection systems

Right panels of antibodies

are essential for these methods.

7070

7171

All of the basic principles used in diagnosis with HE should be performed to IHC practice.

This will lead to less diagnostic errors.

COOPERATION WITH CLINICIANS AND PLANNING STUDIES TOGETHER ESPECIALLY,

WILL BE INCREASED THE EFFECT OF IHC

7272

TEŞEKKÜRLER TEŞEKKÜRLER