Lecture 3 - Chapter 3 Blood Banking Reagents

Chapter 3: Blood Banking - Reagents: Overview and Applications

Objectives

  • U1L3 – O1: Describe the relationship of potency and specificity to blood banking reagents.

  • U1L3 – O2: Compare and contrast polyclonal and monoclonal antibodies.

  • U1L3 – O3: Describe reagents for ABO typing and D typing, and when to run a D control.

  • U1L3 – O4: Describe different types and purposes of reagent red blood cells (RBCs).

  • U1L3 – O5: Describe principles of antiglobulin testing and use of check cells.

  • U1L3 – O6: Distinguish between direct and indirect antiglobulin tests (DATs and IATs) and their indications.

  • U1L3 – O7: Discuss possible errors in antiglobulin testing.

  • U1L3 – O8: Compare polyspecific and monospecific antiglobulin reagents.

  • U1L3 – O9: Describe functions of potentiators in immunohematologic testing.

  • U1L3 – O10: Describe principles of gel technology, microplate techniques, and solid-phase RBC adherence techniques.

Blood Group Antigens and Antibodies

  • Antigens: Embedded structures in RBCs, WBCs, or platelets.

  • Antibodies Form upon exposure to blood group antigens through transfusions or pregnancy. The most concerned classes are IgG and IgM.

    • IgM: Naturally occurring; reacts at room temperature, easily detected.

    • IgG: Immune; requires exposure for detection and needs special serological tests with AHG.

  • Testing includes antibodies in plasma and antigens on cells.

Dynamics of Antigen-Antibody Reactions

  • Forces: Weak, attractive; affected by pH, ionic strength, and temperature.

  • Binding Stages: Dynamic equilibrium represented by a bell-shaped curve (Prozone, Postzone, Zone of equivalence).

  • Union: Dependent on structure and charge of the molecules (lock and key analogy).

  • Interactions are reversible.

Agglutination – Principle and Variables

  • Stages of Agglutination:

    1. Sensitization: Attachment of antibody to antigen on RBCs.

    2. Lattice Formation: Bridges form between sensitized cells.

  • IgG vs IgM Binding:

    • IgG has 2 binding sites.

    • IgM has 10 binding sites, facilitating agglutination.

Variables Affecting Agglutination

  • First Stage Variables:

    • Antigen-Antibody Ratio: Critical for optimal reaction sensitivity.

    • pH: Optimal range of 6.5 to 7.5 for routine procedures.

    • Ionic Strength: Saline neutralizes charges; low-ionic-strength saline (LISS) enhances antibody uptake.

    • Temperature: IgM reacts at room temperature and below; IgG reacts at body temperature.

    • Incubation Time: Time must favor antibody-antigen binding.

Second Stage Variables

  • Immunoglobulin Class Effects: IgG sensitizes but does not agglutinate; IgM easily agglutinates.

  • Electrical Forces: Must overcome repulsive forces; Zeta potential describes maintaining separate RBCs.

Blood Banking Reagents

  • Categories of Reagents:

    • RBCs with known antigens

    • Antisera with known antibodies

    • Antiglobulin reagents (anti-IgG and/or complement)

    • Potentiators to enhance antibody effectiveness.

Reagent Regulation

  • Oversight by the FDA: Commercial blood banking reagents must meet licensing requirements in the Code of Federal Regulations, focusing on specificity and potency.

Quality Control in Blood Banking

  • Purpose of QC: Ensure accuracy and precision of testing procedures and reagents.

    • Criteria for reagent performance, documentation, and corrective actions outlined.

Polyclonal vs Monoclonal Antibodies

  • Polyclonal Antibodies: Sourced from multiple B-cell clones; recognize multiple epitopes (e.g., antihuman globulin - AHG).

  • Monoclonal Antibodies: Derived from a single B-cell clone; recognize a single epitope (techniques such as hybridoma technology used).

Applications of Antiglobulin Tests

  • Direct Antiglobulin Test (DAT): Used to detect in-vivo coated RBCs with globulins, primarily in hemolytic anemia or hemolytic disease of the newborn.

  • Indirect Antiglobulin Test (IAT): Detects in-vitro sensitization; testing for antimicrobials or crossmatching.

Potentiators in Blood Banking

  • Mechanisms: Increase IgG antibody reactivity; use solutions like LISS and BSA.

  • Potentiators: Enhance antibody uptake, thereby increasing testing sensitivity.

Antiglobulin Techniques

  • Two methodologies: Direct and Indirect AHG test, with distinct procedural applications and requirements.

  • AHG Reagents: Monospecific and polyspecific types are important for identifying sensitization and agglutination.

Gel Technology
Gel technology is a modern method used in blood banking for typing and screening. It leverages a gel matrix in microtubes or microplates, allowing for the separation and visualization of agglutination reactions. The gel traps agglutinated cells above a certain density while allowing unagglutinated cells to pass through. This method increases sensitivity and provides clear results due to minimal background interference. Furthermore, it facilitates automation in testing and standardizes results, contributing to improved quality control in blood banking.

Final Review and Practice Questions

  • Summarize content regarding reagent application, errors in testing, and distinguishing between methodologies to reinforce understanding and preparation for testing.