Slide 1

Molecular pathologyDefinition 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 andgenetics, and is sometimes considered a "crossover" discipline. 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.

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 hospitalIn addition to pulmonary complications, other clinical manifestations of H5N1 virus infections may include severe lymphopenia, gastrointestinal symptoms, and liver and renal dysfunctionReactive 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 proinflammatory cytokines and chemokines.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 encephalitisuse 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 culturereal-time RT-PCRIHC and ISHlive 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.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 responseProinflammatory factors. tumor necrosis factor (TNF)related apoptosis-inducing ligand (TRAIL)TNF-

Histopathological featureLung 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 degenerationKidney congested with edemaViral locationImmunohistochemistryImmunohistochemistryorIHCrefers to the process of detecting antigens (e.g., proteins) in cells of a tissue section by exploiting the principle ofantibodiesbinding specifically toantigensinbiological tissues.IHC takes its name from the roots "immuno," in reference to antibodies used in the procedure, and "histo," meaning tissue compare toimmunocytochemistry

Antibodies and Antigens

www.IHCworld.comFab regionParatope + EpitopeAb-Ag complexImmunocytochemistry is performed onsamplesof intact cells that have had most, if not all, of their surroundingextracellular matrixremoved. This includes cells grown within aculture, deposited fromsuspension, or taken from asmear. Immunocytochemistry refers 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 ofbiological tissue, where each cell is surrounded by tissue architecture and other cells normally found in the intact tissuePreparation of AbsPreparation of tissueFixationPresentationChemical preparation1 Ab applicationVisualisation of 1 Ab

How is IHC done?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.Paraformaldehydeis usually used with fixation.Depending on the purpose and the thickness of the experimental sample, either thin (about 4-40m) 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.15Because 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 likeTriton X-100to reducesurface tension, allowing less reagent to be used to achieve better and more even coverage of the samplenonspecific binding causes high background staining that can mask the detection of the target antigenthe samples are incubated with a buffer that blocks the non specific reactive sites to which the primary or secondary antibodies may otherwise bindnormal serum, non-fat dry milk,BSA (bovine serum albumin), or gelatin

Sample labellingThe antibodies used for specific detection can bepolyclonalormonoclonal. 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 mice18B. 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).

antibodies are classified as primary or secondary reagentsPrimary antibodies are raised against an antigen of interest and are typically unconjugatedThe secondary antibody is usually conjugated to a linker molecule, such asbiotin, that then recruits reporter molecules, or the secondary antibody itself is directly bound to the reporter molecule.Direct Method

Tissue AntigenLabeled Antibody21Two-Step Indirect Method

Tissue AntigenPrimary AntibodySecondary Antibody22Staining techniqueAvidin biotin complexInterpretation 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,ApplicationsCancer diagnostics differential diagnosisTreatment of cancer Research 24Part 11. Fixation Fresh unfixed, fixed, or formalin fixation and paraffin embedding2. Sectioning 3. Whole Mount Preparation

Tissue preparation 25Part 21. Antigen retrieval Proteolytic enzyme method and Heat-induced method2. Inhibition of endogenous tissue components 3% H2O2, 0.01% avidin3. Blocking of nonspecific sites 10% normal serum pretreatment26Part 3Make a selection based on the type of specimen, the primary antibody, the degree of sensitivity and the processing time required.staining27Controls 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)28Limitations and Pitfalls of IHC1. The Procedure needs the expertise of the technician as well as the pathologist2.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 100.3.False negative result may be due to :a.Loss of antigen through autolysis b.A scanty amount of tissuec.Extensive necrosis of the tumourd.Inappropriate, de natured antibody4.False positive result may be due to:a.Cross- reactivity of antibodies with other antigensb.The presence of endogenous peroxidase c.Entrapment of normal tissue by tumour cells

31

32In Situ HybridizationIn situhybridization (ISH)is a type ofhybridizationthat uses a labeledcomplementary DNAorRNAstrand (i.e.,probe) to localize a specific DNA or RNA sequence in a portion or section oftissue (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 thestructureof chromosomesFluorescent DNA ISH(FISH) can, for example, be used in medical diagnostics to assess chromosomal integrityRNA ISH (RNAin situhybridization) is used to measure and localize RNAs (mRNAs, lncRNAs and miRNAs) within tissue sections, cells, whole mounts, and circulating tumor cells (CTCs)

Types of ISHRaadioactive ISHSulphur 35Phosphorus 32,33Non radioactive Digoxenin BiotinFluorochromes Method Tissue preparationTake out tissue as soon as possibleFixation of tissue as soon as possibleSectioning : cryostst techniques are usually used. 10 -15 micron size is recommended