Biot 401 Lecture 2: Samples Preparation & Analysis (PDF)

Samples preparation  Degradation of cells and tissues may be due to: u Ischemia u Autolysis u Putrefaction u Other factors in specific organs (e.g., in pancreas, bladder) Ø Ischemia: Term used to describe interval phase between removal of nutrients and ox...

Samples preparation  Degradation of cells and tissues may be due to: u Ischemia u Autolysis u Putrefaction u Other factors in specific organs (e.g., in pancreas, bladder) Ø Ischemia: Term used to describe interval phase between removal of nutrients and oxygen supply, and the cessation of the degradation of the sample after its placed in a preservative or Fixative. Ø Autolysis: PH-induced destruction of cells Ø Putrefaction: external bacterial attack on cells Components are more susceptible to degradative processes u Phosphorylated proteins: fastest components to be degraded u Proteins and mRNA: intermediate u microRNA: more stable than RNA u DNA: the most resilient, suitable for forensic analysis  Cell-based preparations u Standard operating procedures Controlled document that provide a method that must be followed without deviation. Ø Cell culture A method of growing cells outside the body in an environment that enables cells division.  Blood sample Ø Venous blood or whole blood. Ø Used in genomic diagnostic purposes Ø Nucleic acid based biomarkers Ø Genetic disorders u Biomarkers Constituents of cells that are used to measure change occurring in them. They ae frequently used as specific indicators of a disease or to assess its course. u Genetic disorders Changes in the DNA, involving major structural changes , such as the gain or loss of chromosomes, or SNP. When germline, they are inheritable, when sporadic/somatic, they begin in a cell that is capable of division during life.  Types of anticoagulants suitable for use in molecular diagnostics u EDTA (purple tubes): Nucleic acid extraction u Heparin (green tubes): Analysis of WBCs, suitable for DNA extraction, cytogenetic analysis, and immunophenotyping u Sodium citrate (blue tubes): Genetic tests  Genomic analysis of bone marrow u Required for a number of disorders, including myeloma, leukemia, and aplastic anemia. u Requires bone marrow aspiration, then the aspirated samples were collected on suitable anticoagulant.  Cytological analysis u Microscopic observation of individual intact cells u Fixation: A process by which degradation of cells and tissues is irreversibly halted. Optimal fixation result in conservation of cytological and morphological features, as well as the retention of chemical constituents for analysis  Tissue preparations u They can be more challenging than cell preparations u For frozen tissues, degradation is halted u Some analysis using frozen tissues require special care. E.g., examination of frozen preparations of voluntary (skeletal) muscle by microscopy for cytological and enzyme assessment in myopathies, as snap freezing of the biopsy samples in liquid nitrogen alone is not sufficient. So, biopsies should be immersed in isopentane  Tissue preparations u Formalin-fixed paraffin embedding (FFPE): For diagnostic histopathology, formalin fixation is followed by embedding in paraffin wax. The former irreversibly halts degradation of the sample, and the latter provides an internal and external support to the tissues. Molecular analysis Molecular diagnostics can be applied on either: u Intact cells and tissues u Homogenate samples Intact sample assessment u Immunocytochemistry (ICC) u Flow cytometry (FC) u In situ hybridization (ISH) Advantages u Identification of cell types in normal and pathological samples. u Primary tools or as follow-ons after initial cytological or morphological staining. u Valuable diagnostic information. u Valuable contribution to further research into the understanding of diseases process. Immunocytochemistry (ICC)  Immunocytochemistry (ICC) u Technique used for microscopic localization of proteins within and on the surface of intact single cells, and in sections of tissue samples.  Immunocytochemistry (ICC) u In early 1940, the concept of using labelled antibodies for the presence of specific protein in a cellular preparation. u Then, the demonstration of auto-antibodies using frozen section preparations was adopted for diagnostic use. u In 1960, the difficulty of applying ICC to formalin-fixed paraffin embedded (FFPE) tissue sections was described. u Masked epitopes due to the cross-linking effects of formalin fixation in the FFPE process. u So, unmasking of epitope may resolve the problem, facilitate the cytological or morphological diagnosis, as well as targeted therapy in the area of precision medicine. Epitope Retrieval v Proteolytic-induced epitope retrieval (PIER) u Trypsin, pepsin and proteinase K were used to enhance sensitivity by exposing epitopes that have been hidden by the cross-linking effects of formalin. u PIER needs to be carefully controlled. Epitope Retrieval v Heat-induced epitope retrieval (HIER) u Super heating of FFPE tissue sections were used to retrieve epitopes. u HIER employed microwave heating with slides immersed in buffer solutions including metal ions. ICC procedure 1. Epitope retrieval when FFP are used. 2. Blocking non-specific staining 3. Primary antibody incubation 4. Detection of antibody/antigen interaction Blocking Non-Specific Binding u Blocking reactive epitopes and endogenous enzymes prior to primary antibody incubation prevents non- specific binding and mitigates false positive error (incorrect identification of positive signal).  Blocking Non-specific Binding with Serum A. CD14 was detected in paraffin-embedded human tonsil tissue using anti-human CD14 biotinylated affinity-purified polyclonal antibody. B. Non-specific background staining is markedly reduced in a parallel experiment which included a blocking step using animal serum for 15 minutes at room temperature prior to incubation with the primary antibody. Blocking Endogenous Peroxidase u Liver, kidney, and other highly vascularized tissues express high amounts of endogenous peroxidase activity. u These endogenous peroxidase can react with HRP conjugated secondary antibodies used in chromogenic IHC staining, resulting in higher non-specific staining and background levels. u Treating tissues with 3-10% H2O2 prior to incubation with HRP conjugated secondary antibody can quench endogenous peroxidase and significantly reduce non-specific background. Blocking Endogenous Biotin u Liver, kidney, heart, brain, and lung express high amounts of endogenous biotin, which increase non-specific staining in chromogenic IHC staining when a biotinylated secondary antibody is used. u Pre-treating cells with avidin/biotin blocking reagents prior to secondary antibody incubation will reduce non-specific staining due to endogenous biotin. Detection systems  Detection systems Two alternative endpoints are possible with light microscopic ICC: u Fluorescent u Chromogenic Indirect immunofluorescence u Provides ”bright lights” on a dark background u Gave the ability of different fluorophores used for fluorescent labelling to emit light of different wave lengths. u These are useful for the simultaneous localization of multiple antigens.  Chromogenic immunocytochemistry u Based on the use of active enzymes that catalyze substrates to produce insoluble precipitates in the vicinity of the antigen/antibody reaction.  Chromogenic immunocytochemistry u Antigen expression is visualized in chromogenic detection when a soluble substrate is converted by an enzyme to an insoluble colored product that is deposited at the site of antigen expression. u The enzymes horseradish peroxidase (HRP) and alkaline phosphatase are often used in chromogenic detection and function by converting 3,3' diaminobenzidine (DAB) and 3-amino-9-ethylcarbazole (AEC), into brown and red end products, respectively. Applications of ICC u Diagnosis of cancers u Prognostic Markers in Cancer u Drug development to test drug efficacy u Prediction of Response to Therapy u Infectious agents in tissues u Muscle Diseases ICC in HBV diagnosis Scattered hepatocytes show By immunoperoxidase localization, cytoplasmic reactivity with HBcAg can be demonstrated within monoclonal antibody to HBsAg the nuclei or the cytoplasm of using IHC. hepatocytes, or both. ICC in SARS-CoV2 diagnosis Coronavirus antigen-positive pneumocytes and macrophages in lung of a SARS case (Immunoalkaline phosphatase)