Complement Assays Overview

COMPLEMENT ASSAYS

Introduction to Complement Assays

  • Presented by Michael Lacy, PhD, MLS(ASCP)cm.

  • Part of Clinical Immunology 310.

Objectives

  • Understand the objectives of complement assays, similar to those of the complement cascade.

  • Difference in methodology discussions; methods covered later in the lecture.

Testing Complement Components

  • Purpose of testing complement components includes:

    • Measuring specific components as antigens in serum to assess immunological health.

    • Identifying quantitative decreases in complement components potentially indicating deficiencies.

    • Assessing chronic inflammation associated with autoimmune diseases.

    • Evaluating increased susceptibility to infections due to complement deficiencies.

    • Measuring functionality of complement components, where normal levels might still indicate increased susceptibility to infections.

QUANTITATIVE ASSAYS

  • Common Measurements: Includes components such as:

    • C1q

    • C4

    • C3

    • C5

    • Factor B

    • Factor H

    • Factor I

    • C1 inhibitor

  • Example in lab: C3 is commonly measured using Radial Immunodiffusion (RID).

  • Common Methods:

    • Radial Immunodiffusion (RID):

    • Less frequently used due to time consumption.

    • Involves specific antibodies diffused into gel with added antigen.

    • Quantitative measurement.

    • Nephelometry:

    • Rapid method for chemistry analysis; results in minutes.

    • Measures concentration based on light scatter.

    • Limitation: Only provides quantitative values; does not inform on whether components are active.

FUNCTIONAL ASSAYS

  • Emphasis on the classical pathway:

    • Hemolytic Titration (CH50) Assay:

    • Measures how much patient serum is needed to lyse 50% of antibody-sensitized sheep red blood cells (RBCs).

    • Activated by antibodies; comparison to healthy patient values.

    • Requires that the absence of any component (C1-C9) results in an abnormal CH50 value.

    • Disadvantages of CH50:

    • Difficult to perform with no set reference range, requiring individual labs to establish their own standards.

    • Lack of sensitivity; abnormal results do not specify which component is deficient.

Additional Classical Pathway Assays

  • Radial Hemolysis:

    • Rabbit RBCs coated with antibodies implanted into gel.

    • Patient sample added results in lysis of RBCs, producing a clearing around the well.

  • Issue similar to CH50: Determining which component caused the abnormal result.

ELISA in Functional Assays

  • ELISA Methodology:

    • Involves coating wells with IgM or IgG to activate the complement pathway.

    • Addition of patient samples followed by enzyme-labeled antibodies specific for complement components (e.g., anti-C9).

    • Observes color change upon substrate addition if C9 is present, making it a good screening method for components.

Alternative Pathway Functional Assays

  • AH50 Assay (modified CH50):

    • Involves adding MgCl2 and EDTA to chelate calcium, thus blocking classical pathway activation.

    • Utilizes rabbit RBCs as indicators; patient samples compared to controls.

    • Measures differences in serum amounts needed to lyse 50% of RBCs.

  • Other ELISAs:

    • Activation through LPS coating in the well.

    • Detects components C3bBbp or C3bBbC3b.

Complement Activity and Pathway Interactions

  • Importance of recognizing that a patient may have issues with one pathway yet show no symptoms due to compensatory mechanisms through other pathways.

    • Test strips with three areas: Classical coated with IgM, Alternative with LPS, and Lectin with mannose.

Interpreting Decreased Components

  • Different types of decreases:

    • Decreased production (e.g., Liver Disease).

    • Increased consumption (e.g., Autoimmune Disease, Chronic Inflammation).

    • In vitro consumption may cause degradation of components; careful interpretation is crucial.

    • Importance of proper specimen handling to avoid invalid results.

Specimen Handling Guidelines

  • Procedures:

    • Centrifuge blood samples to collect serum.

    • Avoid EDTA, which chelates calcium, complicating analysis.

    • Serum should be frozen or placed on dry ice within 2 hours of collection.

  • Issues Leading to Invalid Results:

    • Inadequate refrigeration, multiple freeze-thaw cycles, prolonged storage, or controls failures can skew results.

Complement Abnormalities

  • Screening should encompass components such as C3, C4, and Factor B, relevant to different pathways.

  • Monitoring inflammation involves measuring C3a, C4a, C5a, and Ba, particularly relevant for conditions such as Rheumatoid Arthritis (RA) and lupus.

Detecting Problems in Complement Function

  • Specific attention to:

    • Impaired Function or Deficiency within Classical, Lectin, and Alternative pathways:

    • C1q, C1r, C1s: low in Classical, normal in others.

    • C4, C2: low in Classical and Lectin pathways, normal in Alternative pathway.

    • Analyzing further for MBL, MASP2 across pathways.

    • Investigating abnormal C3, C5, C6, C7, C8, C9 levels.

  • Acknowledgment of improper specimen handling can lead to misleading results; should remain a concern for upcoming tests.

Complement Fixation

  • Complement used as a reagent in assays to detect antigens or antibodies:

    • Types of antibodies detected include those related to viral, fungal, or rickettsial infections.

Complement Fixation Procedure

  • Two-Step Process:

    1. Introduce unknown antigen or antibody.

    2. Add reagent sheep RBCs coated with hemolysin to indicate lysis presence when complement is available.

Example of Complement Fixation Procedure

  1. Heat inactivate patient complement by heating at 56 °C for 30 minutes.

  2. Perform a dilution series of patient serum with known reagent antigen:

    • If antibodies present, form antibody-antigen complex.

    • Guinea pig complement will bind to the complex (incorporate).

  3. Adding sheep RBCs with hemolysin and incubate for 1 hour; centrifuge and check for hemolysis.

    • Present antibodies result in no lysis as complement is fixed; absence means complement causes lysis.

Controls in Complement Fixation Assays

  • Control Types:

    • Positive/negative controls run parallel to samples.

    • Antigen control examines only antigen addition.

    • Patient serum control analyzes only patient antibody.

    • Cell controls monitor spontaneous lysis without complement.

    • Complement controls verify that complement is functioning correctly through expected lysis outcomes.

Issues Leading to False Results

  • Decreased lysis can indicate inactive complement, incorrect dilutions, or formation of alternative complexes leading to false positives/negatives.

  • Issues identified include fragile RBCs, failure to inactivate complement, inadequate antigen/antibody presence, and potential over-dilution of patient serum.