ASCP - IMMUNOSERO

What are the two main branches of the Immune System?

Adaptive and Innate

Which immune system is described as "Natural, Nonspecific" and is the body's "1st and 2nd Line" of defense?

Innate Immune System

Which immune system is described as "Acquired, Specific" and is the body's "3rd Line" of defense?

Adaptive Immune System

What are some examples of Physical Barriers in the Innate Immune System (1st Line)?

Skin, Mucous membrane, Saliva, Flushing action of urine and tears, Stomach acid, Coughing and sneezing reflexes, Normal flora.
Function: Stop infections before it enters the body.

Name some cellular components of the Bloodborne Innate Immune System (2nd Line).

Neutrophils, Macrophages, Basophils, Eosinophils, Natural killer cells.

Function: Death of dangerous organisms, Direct killing of bacteria.

What is a key component of the Humoral Innate Immune System (2nd Line) that involves a cascade and pathways?

Complement Cascade (Alternative Pathway).

What is Interferon and what is its primary function?

A substance produced by cells (as a result of viral infection) that prevents viral replication.

What are the three main types of T-cells involved in T-cell immunity?

Suppressor T-cells, Helper T-cells, Cytotoxic T-cells.

What is the primary function of Cytotoxic T-cells?

Death of the body's cells that are infected with a virus or otherwise damaged.

After antigen exposure, what do Lymphoblasts differentiate into in B-cell immunity?

Plasma B-cells and Clonal B-cells.

What do Plasma B-cells produce?

Antibodies.

What is the role of Memory B-cells?

To provide long-term immunity by remembering the antigen for future encounters.

What is the major component of the Humoral Adaptive Immunity?

Antibodies.

Which immune system pathway can trigger the Complement Cascade via the Classical Pathway?

The B-cell immunity branch of the Adaptive Immune System (specifically through Antibodies).

What are Acute Phase Reactants?

Humoral components of natural immunity. Their production is driven by IL-6.

What are key characteristics of C-Reactive Protein (CRP)?

• Binds receptors on phagocytic cells (specific, non-specific).

• Capable of opsonization, agglutination, precipitation, and complement activation.

• Binding is non-specific and calcium dependent.

How does CRP levels change during an acute phase response?

Increases rapidly (up to 1000x higher), peaking at 48 hours.

What is the function of Serum Amyloid?

• Activates monocytes and macrophages producing inflammatory molecules.

• Produced in the liver, transported by HDL to the site of infection.

What are the main functions of Complement?

Functions in opsonization, chemotaxis, and lysis of cells.

What is the role of α−1 Antitrypsin?

Limits the harmful effects of the immune system by inhibiting proteases released from leukocytes and regulating expression of proinflammatory cytokines. Note: α−1 Antitrypsin deficiency can lead to pulmonary emphysema and juvenile cirrhosis.

What is the function of Haptoglobin?

Antioxidant, binds irreversibly to free hemoglobin.

What is the primary role of Fibrinogen?

Promotes clot formation.

What is Ceruloplasmin?

Principle copper-transporting protein in plasma.

What is phagocytosis?

Major cellular component of natural (innate) immunity.

What is the first step of Phagocytosis and what makes it possible?

Initiation. Made possible through Toll-like Receptors (TLRs) found on the surface of phagocytes.

What is the second step of Phagocytosis?

Diapedesis. Migration of phagocytes from the blood vessels (capillaries) to injured tissue.

What is the third step of Phagocytosis?

Chemotaxis. Phagocytes move toward or away other cells in response to chemotaxins.

What is the fourth step of Phagocytosis?

Engulfment. Outflowing of cytoplasm occurs and formation of phagosome.

What is the final step of Phagocytosis?

Digestion. Release of enzymes from granules; respiratory burst occurs via oxidative metabolism.

What are the three main types of lymphocytes?

T cells, B cells, and NK cells.

What percentage of lymphocytes in circulation are T cells?

60-80%

What percentage of lymphocytes in circulation are B cells?

10-20%

What percentage of lymphocytes in circulation are NK cells?

10-15%

What type of immunity are T cells primarily involved in?

Cell-mediated immunity.

What type of immunity are B cells primarily involved in?

Humoral-mediated immunity.

What type of immunity are NK cells primarily involved in?

Antibody-Dependent Cellular Cytotoxicity (ADCC).

What do T cells produce?

Cytotoxins (Perforins, Granzymes).

What do B cells produce?

Antibodies.

What do NK cells produce?

Cytotoxins (Perforins, Granzymes).

What do T cells primarily defend against?

Viral, Fungal, Tumors, Graft rejections.

What do B cells primarily defend against?

Bacterial infections.

What do NK cells primarily defend against?

Virus-infected cells, Tumor cells.

What are key surface markers for NK cells?

CD16, IgG receptor, CD56.

Name some mitogens that stimulate T cells.

Phytohemagglutinin, Concanavalin A, Pokeweed.

Name a mitogen that stimulates B cells.

Pokeweed, Staphylococcal Protein A.

What gene rearrangement occurs during B cell maturation?

Rearrangement of genes coding for heavy chains (Chromosome 14) and light chains (Chromosome 2 or 22).

What are the key markers for a Pro-B cell?

CD19, CD10, CD45, CD24, CD22.

What happens during the Pre-B cell stage (Precursor)?

Appearance of mu chains (μ) and heavy chain.

What are the characteristics of an Immature B cell (Competent B cell)?

Selection process, appearance of surface IgM, CD21, CD40, MHC Class II.

What is the key characteristic of a Mature B cell?

Appearance of surface IgM and IgD, MHC Class II.

What is a characteristic of an Activated B cell?

Appearance of CD25 which serves as receptor for IL-2.

What is the primary function of a Plasma cell?

Production of antibodies.

What is a characteristic of a Lymphoma cell?

Cytoplasmic Immunoglobulin.

What gene rearrangement occurs during T cell maturation?

Rearrangement of genes coding for T-cell receptor.

What are the markers for a Double Negative Thymocyte?

(-) CD4 and CD8. Additional: (+) CD2, CD5, and CD7.

What are the markers for a Double Positive Thymocyte?

(+) CD3, CD4, and CD8. This stage involves the "Selection Process."

What is the key characteristic of a Mature T cell?

Either CD4 or CD8 is present (but not both).

What is a characteristic of an Activated T cell?

Appearance of CD25 which serves as receptor for IL-2.

What is flow cytometry primarily used for in lymphocyte identification?

Rapid and convenient technique for generating immunophenotypic data.

What clinical conditions is flow cytometry useful for evaluating and prognosticating?

Leukemia, lymphoma, and immunodeficiency.

What is the fundamental principle behind flow cytometry?

Based on Light Scattering derived from suspension of particles of a flowing stream of fluid.

What does Forward Scatter (FSC) measure in flow cytometry?

Cell size.

What does Side Light Scatter (SSC) measure in flow cytometry?

Cell internal complexity / granularity.

What is the purpose of fluorescent antibodies in flow cytometry?

Quantifying cell subpopulation or cell components.

Name some common fluorophores used in flow cytometry.

FITC, TRITC, Phycoerythrin.

What types of specimens can be used for flow cytometry?

Blood / Bone Marrow (BM). Other options include body fluid aspirates and tissue samples.

What anticoagulants are commonly used for flow cytometry specimens?

EDTA, Heparin, Acid citrate dextrose.

What is the recommended stability time for blood samples collected with EDTA for flow cytometry?

30 minutes. (EDTA is the anticoagulant of choice for blood samples).

What is the recommended stability time for samples collected with Heparin, and what does it improve?

24 hours. Heparin improves the stability of the sample.

Is removal of Red Blood Cells (RBCs) required prior to flow cytometry testing?

Yes, removal of RBCs is required.

How can RBCs be removed using density gradient centrifugation?

Using Ficoll-Hypaque solution.

What are the layers produced by density gradient centrifugation and their implications?

• Plasma (top) - time consuming.

• Mononuclear cells (middle) - can result in relative loss of some cell populations.

• RBCs, granulocytes (bottom).

What is the preferred method for RBC removal using a lysing agent?

Uses ammonium chloride (preferred method).

What is the HLA locus and where is it located?

Composed of >200 genes located at the short arm of Chromosome 6.

How are HLA antigens inherited?

Inherited as a haplotype from each parent in a simple Mendelian inheritance pattern. There's a 25% chance that two siblings will be HLA identical.

What are the major genes for HLA Class I?

HLA-A, B, C.

What are the major genes for HLA Class II?

HLA-DP, DQ, DR.

What are the minor genes for HLA Class I?

HLA-E, F, G.

What are the minor genes for HLA Class II?

HLA-DM, DO.

What are the characteristics of HLA Class III?

Secreted proteins with immune function, not expressed on cell surfaces. Includes Complement proteins and cytokines (C2, C4, C4b, CR, B, TNF).

Describe the structure of HLA Class I molecules.

3 α (alpha) chains + β (beta) microglobulin.

Describe the structure of HLA Class II molecules.

2 α (alpha) chains + 2 β (beta) chains.

FLASHCARD: HLA Class I

📍 Cell Distribution:
All nucleated cells

🧱 Peptide Binding Cleft:
Closed at both ends

🔗 Peptide Size Bound:
8–11 amino acids

🎯 T Cell Interaction:
Presents endogenous antigens to CD8+ T cells

🧬 Associated Alleles:

• B27 – Ankylosing Spondylitis

• B5 – Behçet Disease

• B*57:01 – Abacavir sensitivity

• B*15:02 – Carbamazepine sensitivity

HLA Class II

📍 Cell Distribution:
Antigen-presenting cells (APCs):
Macrophages, Monocytes, B cells, Dendritic cells, Thymic epithelial cells

🧱 Peptide Binding Cleft:
Open at both ends

🔗 Peptide Size Bound:
13–18 amino acids

🎯 T Cell Interaction:
Presents exogenous antigens to CD4+ T cells

🧬 Associated Alleles:

• DQ2, DQ8 – Celiac Disease

• DQ B1*06:02 – Narcolepsy

• DR B1*04 – Rheumatoid Arthritis

• DR3 – Graves’ Disease, Hashimoto’s Thyroiditis

• DR5 – Myxedema, Atrophic Thyroiditis

HLA Functions & Applications

#1 – Immune Response:
Mounting adaptive immune responses

#2 – Histocompatibility:
Used for histocompatibility matching (e.g., transplant), paternity testing

#3 – Disease Association:
HLA alleles are markers for autoimmune, hypersensitivity, and infectious disease predisposition

Immunoglobulin Structure 1

🔗 Basic Structure:
2 identical Heavy chains + 2 identical Light chains (tetrapeptide)

🧬 Heavy Chain Types:
μ (Mu), γ (Gamma), α (Alpha), δ (Delta), ϵ (Epsilon)

🧬 Light Chain Types:
Kappa (κ): 65–70%
Lambda (λ): 30–35%

🧷 Chain Linkage:
Held together by noncovalent forces and disulfide bridges (H-H, H-L)

Immunoglobulin Structure 2

🔬 Chain Regions:
Each chain has a variable region and one or more constant regions

🧪 Variable Region Detail:
First 110 amino acids

🧮 Hinge Region:
Between CH1 and CH2, rich in proline

🧲 CH2 Receptors Bind:
Complement proteins

🧲 CH3 Receptors Bind:
Phagocytes

Complement System Overview

🧩 Definition:

Complex system of >30 soluble and membrane-bound proteins that mediate and enhance host defense.

🔥 Stability:
Heat-labile (not stable in heat); found in non-immune serum

🏭 Synthesis Site:
Mostly in the liver

🧪 Exceptions:

• C1: Intestinal epithelial cells

• Factor D: Adipose tissue

🧫 Precursors:
Mostly synthesized as inactive zymogens, activated in specific sequence

Classical Pathway

• Trigger: Ag-Ab complex (2× IgG or 1× IgM)

• Sequence: C1 → C4 → C2 → C3 → C5 → C6 → C7 → C8 → C9

• C3 Convertase: C4b2a

• C5 Convertase: C4b2a3b

MBL (Lectin) Pathway

• Trigger: Mannose binding

• Sequence: MASP → C4 → C2 → C3 → C5 → C6 → C7 → C8 → C9

• C3 Convertase: C4b2a

• C5 Convertase: C4b2a3b

Alternative Pathway

• Trigger: Bacterial LPS, IgA, or other pathogen surfaces

• Sequence: C3 → C5 → C6 → C7 → C8 → C9

• C3 Convertase: C3bBb

• C5 Convertase: C3bBb3b

IgG

• Form in Blood: Monomer

• Concentration: 120 mg/dL (Highest)

• Complement Fixation: Yes

• Key Property: Crosses placenta

IgM

• Form in Blood: Pentamer

• Concentration: 15 mg/dL

• Complement Fixation: Yes

• Key Properties: First Ig made (fetus & naive B cells), hemolytic, B cell receptor

IgA

• Form in Blood: Monomer (blood), Dimer (secretions)

• Concentration: 30 mg/dL

• Complement Fixation: Yes (Alternative pathway)

• Key Properties: Most abundant in secretions (saliva, tears, etc.)

IgD

• Form in Blood: Bound to immature B cells

• Complement Fixation: No

• Key Role: B cell development

IgE

• Form in Blood: Bound to mast cells

• Complement Fixation: No

• Key Role: Allergies and parasitic infection

Isotype

Definition: Based on type of heavy chain → IgG, IgA, IgM, IgD, IgE

Allotype

Definition: Minor variations in constant regions (individual differences)

Idiotype

Definition: Unique antigen-binding site, formed by variable regions of H and L chains

Anti-idiotype

Definition: Antibody that recognizes an idiotype as an antigen