MLT 115 Precipitation and Aggulation

Overview

  • The instructor expresses happiness with the students’ progress.

  • The second exam is tentatively scheduled for February 10, but adjustments might be made due to scheduling and student availability.

Exam Schedule and Details

  • Exam Two Date: February 5.

    • Topic: Cellular immunity and complement.

  • Exam Content Overview:

    • Key questions previously shared by the instructor:

    • Name three characteristics of B lymphocytes.

    • Name three characteristics of T lymphocytes (with extra credit options).

  • Hints for Preparation: The instructor reassures that students have sufficient time for review and preparation for the exam.

Review and Examination Preparation

  • Encouragement to embrace the review session.

  • Study Focus Areas include:

    • Knowing the sequence of steps in certain processes, without needing to define them.

  • Steps of Phagocytosis:

    1. Pneumocaxis.

    2. Adherence.

    3. Engulfment.

    4. Lysis.

    5. Inflammation.

Complement System

  • Mention of a handout distributed detailing essential information about the complement system.

  • Test questions derived from this handout will be critical for Exam Two.

  • Important Note: No requirement to draw the complement cascade on the exam.

Cellular Immunity

  • Discussion on primary and secondary lymphatic tissue locations, lymph nodes, and spleen functions:

    • White Pulp and Red Pulp in the spleen are emphasized.

  • Characteristics of T lymphocytes:

    • Longevity: T lymphocytes can survive for extended periods, whereas B lymphocytes do not.

    • Cloning Capability: B lymphocytes can generate clones to produce specific antibodies.

  • Important T lymphocyte markers:

    • Helper T Cells: CD markers are CD3+, CD4+, and CD8-.

    • Cytotoxic T Cells: CD markers are CD3+, CD4-, CD8+.

Phagocytosis and Immune Response

  • Overview of the immune response cascade:

    • Activation of Neutrophils as the first line of defense in case of broken skin.

    • Importance of segmented neutrophils in immediate immune responses.

  • **Cell Types Discussed: **

    • Natural Killer (NK) cells and their role in cellular immunity.

*Methodology Updates

  • The instructor assures that methodology reviews will be lighter than previous sessions to avoid redundancy.

  • Specific methodologies to be discussed:

    • Agglutination and Precipitation Techniques: New material for this course.

Precipitation

  • Definition: The aggregation of soluble test antigens upon interacting with antibodies. This is an unlabeled immunoassay.

  • The antigen and antibody are in a solution and then they become a solid after a bond is created

  • Measurement techniques:

    • Historical methods involve diffusion through agar or gel.

    • Example of zone formation indicating antigen-antibody bonding.

  • Diagram references to observe precipitation in practical scenarios.

  • The Two types of testing methods we usually use: Precipitation and Agglutination. These produce a visible expression of the aggregation of antigens and antibodies.

  • Agglutination: The process whereby specific antigens (e.g. red blood cells) aggregate to form larger visible clumps when the corresponding specific antigen is present in the serum.

Radial Immunodiffusion

  • This measures diameter of the zone of precipitation, which is the place where the antigen and antibody bond is causing precipitation to diffuse around the well.

  • A patient’s specimen is in the middle circle of a gel plate, while there are five circles around it that are standard speciments with known antigen concentrations.

  • The diameter of the zone of precipitation is equally proportional to the concentration of the antigen. e.g. the greater the concentration the greater the zone of precipitation

  • The process of using the flow chart to find the patient’s antigen concentration: The diameter of the standard’s zones of precipitation are measured and plotted on a flow chart. Then we create a curve. Then we measure the patient’s diameter of zone of precipitation against the standards. Then we calculate the patient’s antigen concentration through the standard’s given concentration, just like how we measure and find the Absorbance and Concentration of an unknown through Beer’s Law.

  • Similar in principle to Ouchterlony diffusion (found in pg. 133 in the book where the known antigen is placed beside the known antibody on a gel plate, and if the bond exists, creates a precipitating line called the “line of identity), except the known antibody is uniformly distributed in the gel medium.


Flocculation Tests

  • These are used for antibody detection based on the interaction of soluble antigen with an antibody forming a precipitation of fine particles

  • There are 2 of these tests: VDRL and RPR

  • VDRL (Venereal Disease Research Laboratory): This is only used on CSF specimens when we suspect Teriary Syphilis or neurosyphilis in a patient.

  • RPR (Rapid Plasmin Reagin): This test uses a reagin, which is not an antibody. It is a substance produced in the plasma of patients with syphilis.

  • RPR tests causes many false positives.

VRDL

  • The antigen is Cardrolipin lecithin-coated cholesterol particles.

  • The antibody is a protein called Reagin

  • In a solution, if the Reagin is present, then both the antibody and antigen will form cholesterol precipitates, which can be viewed microscopically

RPR

  • Similar to VRDL, Antigen is Cardiolipin Lecithin-coated charcoal particles

  • The patient’s plasma could possibly have reagin in them

  • This is similar to VRDL but with Charcoal particls so that it can be seen macroscopically

  • If this test is negative, the patient doesn’t have syphilis. If positive, then we have to do the VDRL. If the VDRL is positive, we have to do a fluorescent technique to look for the syphilis itself.

  • This test is being set aside for VDRL. VDRL is the better test to use to detect syphilis.


Latex Agglutination Testing

  • Definition: Utilizes soluble antigens attached to latex particles to detect antibodies in serum samples. This is a rapid test but not a point-of-care test, and it is not waived by CLIA.

  • Process: Antigen is attached to the latex. Then, add the patient’s plasma. If the antibody is present, agglutination happens (or clumping)

  • This process can be done with either an antigen attached to the latex or the antibody attached to the latex and the antigen is possibly found in the patient’s plasma.

  • Classification of latex agglutination as rapid testing, but not classified as point of care due to complexity requirements.

  • Distinction between IgM and IgG during testing phases.

    • Key Note: Presence of IgM indicates current illness, while IgG indicates recovery.

Immune Testing Methods

  • Overview of different assays: ELISA, agglutination tests, and their correlates in clinical practices.

  • Importance of understanding patient’s antibody response for effective diagnosis and management.

  • Techniques fostering effective differentiation of antibodies present in diagnostic blood tests.

Quality Control

  • Quality assurance and control discussions will occur in future sessions.

Study Recommendations

  • Emphasis on reading primary texts for clarity.

  • Recommendations to focus on key concepts outlined and revisable content, avoiding exhaustive textbook readings.

  • Queries for clarification encouraged at every class meeting.

  • Next Session Goals: Focus will shift to diseases planning and further detailed notes regarding methodologies in upcoming exams.