Molecular pathology

1. Molecular pathology

2. Definition • Molecular pathology • is the study and diagnosis of disease through the examination of molecules within organs, tissues or bodily fluids.  • Molecular pathology shares some aspects of practice with both anatomic pathology and clinical pathology, molecularbiology, biochemistry, proteomics and genetics, and is sometimes considered a "crossover" discipline.

3. It is multi-disciplinary in nature and focuses mainly on the sub-microscopic aspects of disease. • development of molecular and genetic approaches to the diagnosis and classification of human diseases, • the design and validation of predictive biomarkers for treatment response and disease progression, • the susceptibility of individuals of different genetic constitution to develop disorders.

4. Human H5N1 disease is clinically and pathologically distinct from that caused by seasonal human influenza A H3N2 or H1N1 viruses. • a rapid progression of lower respiratory tract disease, often requiring mechanical ventilation within days of admission to a hospital • In addition to pulmonary complications, other clinical manifestations of H5N1 virus infections may include severe lymphopenia, gastrointestinal symptoms, and liver and renal dysfunction

5. Reactive hemophagocytosis in multiple organs, and occasional detection of viral antigen or viral RNA in extrapulmonary organs suggest a broader tissue distribution of H5N1 viruses compared with seasonal viruses in fatal human cases. • Patients with severe H5N1 disease have unusually higher serum concentrations of proinflammatorycytokines and chemokines.

6. The elevation of plasma cytokine levels was positively correlated with pharyngeal viral load and may simply reflect more extensive viral replication. • Although H5N1 virus infection of humans is primarily one of the lower respiratory tract, in rare, severe cases, disseminate beyond the lungs and infect brain, intestines and lymphoid tissues and result in extra-pulmonary clinical manifestations including encephalopathy or encephalitis

7. use of different techniques to detect virus distribution and infection of 5 organ systems in a laboratory confirmed fatal human H5N1 virus infection, and analyze the relationship between viral load in tissues and host response. • Virus distribution: • Virus culture • real-time RT-PCR • IHC and • ISH

8. live virus was recovered from respiratory tissues including lung, trachea, bronchus and aortopulmonary vessel. In the digestive system, virus was isolated from tissues collected from the ileum, colon and rectum, but not the stomach, duodenum or liver. • virus culture was also positive on tissues collected from brains, ureter and axillary lymph-node. Sequencing results showed that the sequences of isolates are identical.

9. The tissue distribution of viral RNA or antigen detected by ISH and IHC stains respectively, was also generally consistent with virus isolation by culture or real time RT-PCR result. • Viral load is associated with host response • Proinflammatory factors. • tumor necrosis factor (TNF)–related apoptosis-inducing ligand (TRAIL) • TNF-α

10. Histopathological feature • Lung showed diffused alveolar damages including intraalveolar edema. • focal intra-alveolar hemorrhage. • necrosis of alveolar line cells, • focal desquamation of pneumocytes in alveolar spaces, • interstitial mononuclear inflammatory cell infiltrates, and extensive hyaline membranes. • Liver congested with edema and focal fatty degeneration • Kidney congested with edema

11. Viral location Immunohistochemistry Immunohistochemistry or IHC refers to the process of detecting antigens (e.g., proteins) in cells of a tissue section by exploiting the principle of antibodies binding specifically to antigens in biological tissues. IHC takes its name from the roots "immuno," in reference to antibodies used in the procedure, and "histo," meaning tissue compare to immunocytochemistry

12. Paratope + Epitope Ab-Ag complex Antibodies and Antigens Fab region www.IHCworld.com

13. Immunocytochemistry is performed on samples of intact cells that have had most, if not all, of their surrounding extracellular matrix removed. This includes cells grown within a culture, deposited from suspension, or taken from a smear. • Immunocytochemistryrefers to localization in isolated cells or labelling directed to cell specific compartment (e.g. the cell membrane, Golgi or lysosomes, etc.) • In contrast, immunohistochemical samples are sections of biological tissue, where each cell is surrounded by tissue architecture and other cells normally found in the intact tissue

14. How is IHC done? • Preparation of Ab’s • Preparation of tissue • Fixation • Presentation • Chemical preparation • 1° Ab application • Visualisation of 1° Ab

15. Fixation • complete preparation of the sample is critical to maintain cell morphology, tissue architecture and the antigenicity of target epitopes. This requires proper tissue collection, fixation and sectioning.  • Paraformaldehyde is usually used with fixation. • Depending on the purpose and the thickness of the experimental sample, either thin (about 4-40 μm) sections are sliced from the tissue of interest, or if the tissue is not very thick and is penetrable its used whole. •  slices are mounted on slides.

16. Because of the method of fixation and tissue preservation, the sample may require additional steps to make the epitopes available for antibody binding, including deparaffinization and antigen retrieval. • (microwave method, enzyme method, hot incubation method) • Detergents like Triton X-100 to reduce surface tension, allowing less reagent to be used to achieve better and more even coverage of the sample  • nonspecific binding causes high background staining that can mask the detection of the target antigen

17.  the samples are incubated with a buffer that blocks the non specific reactive sites to which the primary or secondary antibodies may otherwise bind • normal serum, non-fat dry milk, BSA (bovine serum albumin), or gelatin

18. Sample labelling • The antibodies used for specific detection can be polyclonal or monoclonal. • Polyclonal antibodies: Large complex antigens may have multiple epitopes and elicit several antibody types. Mixtures of different antibodies to a single antigen are called polyclonal antibodies. Produced by injecting a specific antigen into a rabbit and taking its serum. • Monoclonal antibodies: Antibodies specific for a single epitope and produced by a single clone are called monoclonal antibodies and are commonly raised in mice

19. B. Labeling Antibodies: • Antibodies are not visible with standard microscopy and must be labeled in a manner that does not interfere with their binding specificity. • Common labels include fluorochromes (eg, fluorescein, rhodamine), enzymes demonstrable via enzyme histochemical techniques (eg, peroxidase, alkaline phosphatase), and electron-scattering compounds for use in electron microscopy (eg, ferritin, colloidal gold).

20. antibodies are classified as primary or secondaryreagents • Primary antibodies are raised against an antigen of interest and are typically unconjugated •  The secondary antibody is usually conjugated to a linker molecule, such as biotin, that then recruits reporter molecules, or the secondary antibody itself is directly bound to the reporter molecule.

21. Direct Method Labeled Antibody Tissue Antigen

22. Two-Step Indirect Method Secondary Antibody Primary Antibody Tissue Antigen

23. Staining technique • Avidin –biotin complex • Interpretation of Results: • Results are interpreted in lights of the appropriate staining of negative and positive controls. A Positive reaction is indicated by brown staining at a specific site of cellular antigen,

24. Applications • Cancer diagnostics • differential diagnosis • Treatment of cancer • Research

25. Part 1 Tissue preparation 1. Fixation Fresh unfixed, fixed, or formalin fixation and paraffin embedding 2. Sectioning 3.Whole Mount Preparation

26. 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

27. Part 3 staining • Make a selection based on the type of specimen, the primary antibody, the degree of sensitivity and the processing time required.

28. 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.(known tissue) • Negative Control It is to test for the specificity of the antibody involved.(non-immune serum)

29. Limitations and Pitfalls of IHC 1. The Procedure needs the expertise of the technician as well as the pathologist 2. Certain markers initially thoughts to be specific for certain tissues or tumors have proved to be shared by several other tissues and other neoplasm,eg S 100.

30. 3. False negative result may be due to : a. Loss of antigen through autolysis b. A scanty amount of tissue c. Extensive necrosis of the tumour d. Inappropriate, de natured antibody 4. False positive result may be due to: a. Cross- reactivity of antibodies with other antigens b. The presence of endogenous peroxidase c. Entrapment of normal tissue by tumour cells

33. In Situ Hybridization • In situ hybridization (ISH) is a type of hybridization that uses a labeled complementary DNA or RNA strand (i.e., probe) to localize a specific DNA or RNA sequence in a portion or section of tissue (in situ), or, if the tissue is small enough (e.g. plant seeds, Drosophila embryos), in the entire tissue (whole mount ISH), in cells and in circulating tumor cells (CTCs). • DNA ISH can be used to determine the structure of chromosomes

34. Fluorescent DNA ISH (FISH) can, for example, be used in medical diagnostics to assess chromosomal integrity • RNA ISH (RNA in situ hybridization) is used to measure and localize RNAs (mRNAs, lncRNAs and miRNAs) within tissue sections, cells, whole mounts, and circulating tumor cells (CTCs)

36. Types of ISH • Raadioactive ISH • Sulphur 35 • Phosphorus 32,33 • Non radioactive • Digoxenin • Biotin • Fluorochromes

37. Method • Tissue preparation • Take out tissue as soon as possible • Fixation of tissue as soon as possible • Sectioning : cryostst techniques are usually used. 10 -15 micron size is recommended