Immunology and Molecular Genetics

What is clinical immunology?

Dealing with the study, diagnosis and management of patients with diseases resulting from disordered immunological mechanism

What do clinical immunology laboratories do?

- Clinical immunology laboratories specialize in the evaluation of autoimmune disease, immunodeficiencies, immunoproliferative disorders, allergy, and some aspects of infectious disease serology

- Investigates novel aspects of the cellular immune response to pathogens

- The laboratory performs and offers expert interpretation on a board array of laboratory tests and comprehensive consultation in clinical and diagnostic immunology

What is the immune system?

- A complex system of tissues, cells, cell products, and biology active chemicals

- Produces an immune response

- Defence mechanism against foreign substances called antigens (ag)

What is natural response?

- Includes physical barriers (eg. skin, mucous membranes, etc), white blood cells like neutrophils, and proteins that cause inflammation

- Non-specific

- Does not require exposure to an antigen

What is specific immunity?

- Recognition : Recognizes foreign antigens and distinguishes them from "self"

- Specificity : Reacts with a specific antigen without reacting with others

- Memory : Anamnestic response; a renewed rapid production of an antibody following second or later exposure with the provoking antigen or with related antigen

What are the primary lymphoid organs where lymphocytes are produced?

Bone marrow

What type of cells are produced in the bone marrow?

Lymphocytes (white blood cells)

What are the two main types of lymphocytes?

B-cells and T-cells

What are secondary lymphoid organs?

Lymph nodes, spleen, tonsils, and certain tissues in mucous membranes

What is the function of secondary lymphoid organs?

They are where the immune system cells fight off germs and foreign substances

What is humoral immunity?

The process of adaptive immunity manifested by the production of antibodies by B lymphocytes

What does humoral immunity protect against?

Bacteria, toxins, and circulating antigens

What is cell-mediated immunity?

T lymphocytes protect against viruses, fungi, tumor cells, and intracellular organisms

What are immunoglobulins (Ig)?

Glycoprotein molecules produced by plasma cells

What is the function of antibodies (ab)?

They recognize and bind to specific antigens, such as bacteria or viruses, and aid in their destruction

What occurs during the primary immune response?

It occurs after the first exposure to an antigen

What is the first antibody produced during the primary immune response?

Mainly IgM, although small amounts of IgG are also produced

How does the amount of antibody produced in the primary immune response vary?

It depends on the nature of the antigens; usually produced in low amounts and declines rapidly

What is the affinity of antibodies for antigens during the primary immune response?

The affinity of antibody for antigen is low

What type of cell response occurs during the primary immune response?

Naive B and T-cell response

What is produced as a result of the primary immune response?

Memory cells with high specificity to the exposed antigen

What happens during the secondary immune response?

Lymphocytes remember the antigen, and memory cells respond

Why are immunizations effective in the secondary immune response?

Because memory cells respond to the antigen

What type of antibodies are mainly produced during the secondary immune response?

Mainly IgG antibodies, although sometimes small amounts of IgM are produced

How does the quantity of antibodies produced in the secondary immune response compare to the primary response?

Usually 100-1,000 times more antibodies are produced, and levels remain high for a longer period of time

What is the affinity of antibodies for antigens during the secondary immune response?

The affinity for antigen is high

What is the basis of the immunology lab?

Knowing the pattern of antibody response

What do assays in immunology labs use?

Assays are developed based on the disease, bacteria, or virus target.

Autoimmune Disease

- Rheumatoid arthritis

- Lupus

- Juvenile Type I Diabetes

- Myasthenia gravis

Hypersensitivities

- Hay Fever

- Astma

- Dermatitis

Malignancies

- Lymphoma

- Leukemia

- Multiple myeloma

Acquired Immunodeficiencies

- Infections

- Systemic Disease

- Malignancies

- Drug reactions

- Irradiation

Congenital Immunodeficiencies

- DiGeorge syndrome

- Agammaglobulinemia

- SCID (Severe Combined Immune Deficiency

What are tests of immune function based on?

Antigen-Antibody Reactions

What type of results can tests of immune function yield?

Qualitative (positive or negative) and quantitative

What does the term 'titer' refer to in immunological tests?

Reciprocal of the highest dilution of patient's serum showing a positive reaction with antigen

What is agglutination in the context of immunological tests?

Visible clumping of cells or particles due to their reaction with an antibody

What is agglutination inhibition?

A test that measures the ability of antibodies to prevent agglutination of cells or particles

What is agglutination?

Agglutination is the process where antibodies cause the cross-linking of particulate antigens, usually found on a cell

What is direct agglutination?

Direct agglutination tests patient serum against large cellular antigens to screen for the presence of antibodies

What is a characteristic of direct agglutination?

In direct agglutination, the antigen is naturally present on the surface of cells

What occurs during the Ag-Ab reaction in direct agglutination?

The Ag-Ab reaction forms directly visible agglutination

What is indirect agglutination?

Indirect agglutination involves attaching antibodies to inert latex beads to improve visualization of agglutination

What does the agglutination of beads indicate in indirect agglutination?

The agglutination of the beads is a marker for antibody binding to some other antigen

What can indirect assays detect?

Indirect assays can be used to detect the presence of either antibodies or specific antigens

What is the role of antibodies in neutralization tests?

The presence of antibodies prevents the antigen from functioning properly

What do neutralization tests measure?

Neutralization tests are based on the binding of antibodies to the pathogen or to toxins

What can neutralization tests be applied to detect?

Neutralization tests can detect pathogens or toxins and identify antibodies

What are neutralizing antibodies (NAbs)?

Neutralizing antibodies (NAbs) play a vital role in the antiviral response

How do neutralizing antibodies prevent viral entry?

They block receptor engagement, arrest the membrane fusion process, and accelerate the decay of viral particles

What pathogens or toxins can neutralization tests be used to detect?

Neutralization tests can detect streptolysin or rabies

What is one application of neutralization tests in vaccines?

They can monitor the neutralizing effect of vaccines

What is the process of Complement Fixation?

Red blood cells are artificially coated with the desired antigen and then mixed with patient serum

What happens when patient antibodies bind to the antigen on red blood cells in Complement Fixation?

The binding activates complement and the red cells are lysed

What are Labeled Methods?

Laboratory techniques used to detect and measure specific antigens or antibodies by attaching a detectable tag to them

What are the components of a Labeled Assay?

- Analyte : The target molecule being detected (can be labeled or unlabeled).

- Specific Antibody : Binds to the antigen of interest.

- Separation of Bound and Free Components : Ensures accuracy by removing unbound labels.

- Detection System : Measures the label (e.g., fluorescence, color change).

- Standards/Calibrator : Provides a reference for interpreting results

What is the purpose of an antibody titration?

An antibody titration can help determine antibody concentration levels

How are antibody titers determined?

Two-fold serial dilutions of serum containing an antibody are made, then tested against cells possessing the target antigen

What is the titer in antibody testing?

The titer is the reciprocal of the greatest dilution in which agglutination is observed

What are Titer Results and their significance?

- Titers provide more value when tested in parallel with a previous titer specimen.

- A comparison of current and previous specimens should be made.

- A change in titer of 2 or more tubes is considered significant.

Why is a Titer performed?

- To assess acute and convalescent stages of infection.

- For prenatal (Rh) testing

- To verify past infections

- To confirm vaccination status

What are some pathogen tests in immunology?

- Rubella

- Syphilis

- Lyme

- Hepatitis A (outbreak situations)

- Hepatitis B

- Hepatitis C

- HIV

- Chlamydia

- Gonorrhea

What if Flow Cytometry?

- A technique to detect and measure physical and chemical characteristics of cells or particles.

- Analyzes 13 parameters (forward scatter, side scatter, 11 immunofluorescent colors)

- Relies on fluidics, optics, and electronics

- Light scatter depends on cell structure, size, and shape

What are six common uses for Flow Cytometry?

- Counting CD4 T-cells in HIV patients

- Counting reticulocytes to assess anemia or bone marrow health

- HLA testing for organ transplant compatibility

- Counting sperm for infertility or vasectomy verification

- Diagnosing leukemia/lymphoma

- Assessing platelet functionality

What does a Molecular Genetic Technologist do?

- Analyzes DNA/RNA for cancer, bacterial/viral infections, and inherited disorders

- Extracts DNA/RNA from blood or tissues

- Performs molecular assays and maintains quality control

- Reviews results for accuracy

What are the steps for Nucleic Acid Extraction?

1. Cell lysis

2. Protein removal

3. Precipitation (using EtOH) or binding to a solid column

4. Washing

5. Elution or purified DNA/RNA

How is DNA/RNA analyzed?

- Gel Electrophoresis : Separates DNA fragments

- Sequencing : Identifies DNA bases (A, T, G, C)

- PCR : Amplifies DNA for analysis

- Hybridization : Detects specific sequences using labeled probes

What is PCR (Polymerase Chain Reaction)?

- Amplifies DNA segments using thermal cyclers

- Aids in detecting pathogens and genetic abnormalities

- Evaluated using capillary electrophoresis instrumentation

What is DNA Sequencing?

- Determines the order of DNA bases (A, T, G, C)

- Identifies mutations that may lead to diseases like cancer or cystic fibrosis

What is Hybridization?

- Detects specific DNA sequences using a labeled probe

How can Hybridization be applied?

- Antibiotic resistance genes

- Mutations (causing mismatches in DNA)

What are the applications of nucleic acid-based tests?

- Detect unique bacterial, fungal, or viral sequences

- Identify defective genes in inherited diseases (e.g., cystic fibrosis)

- Detect cancer mutations or chromosomal translocations

What is Virology?

The study and identification of pathogenic viruses

What are the four modes of viral transmission?

1. Enteric : Fecal-oral route (e.g., Rotavirus, Hep A)

2. Respiratory : Droplets (e.g., Influenza, Measles)

3. Zoonotic : Animal-human transmission (e.g., Rabies, West Nile)

4. Sexual : Direct contact (e.g., HIV, Herpes Simplex Virus-2)

What are methods of virus detection?

- Tissue Culture, bird embryos, live mammals

- Electron Microscopy : Visualizes viruses

- Antibody Detection: IgM/IgG testing for Hep A/B/EBV

- ELISA: Detects viral proteins via enzyme-substrate reactions

- Western Blotting: Separates viral proteins using electrophoresis

- PCR: Detects DNA viruses

- RT-PCR: Detects RNA viruses