THE COMPLETE OVERVIEW OF COMPLEMENT FIXATION TESTS IN MEDICAL DIAGNOSTICS

The Complement Fixation Test (CFT) is a classical serological technique widely employed in medical diagnostics to detect the presence of specific antibodies or antigens in a patient’s serum. Its principle relies on the ability of antigen-antibody complexes to bind and activate the complement system, leading to the “fixation” or consumption of complement components.

Understanding the Complement Fixation Test (CFT)

The Complement Fixation Test is a two-stage immunological assay primarily used for the diagnosis of infections (bacterial, viral, fungal, parasitic) and certain autoimmune diseases. It is an indirect test, meaning it does not directly visualize the antigen-antibody reaction, but rather its consequence – the utilization of complement.

Principle: The core principle of CFT revolves around the consumption of complement. When an antibody (if present in a patient’s serum) binds to its specific antigen, it forms an immune complex. This immune complex possesses sites that can bind and activate the classical pathway of complement. If complement is ‘fixed’ or consumed by this primary antigen-antibody reaction, it is no longer available to participate in a subsequent indicator reaction.

The test proceeds in two distinct stages:

• Primary (Fixation) Stage: The test involves incubating the patient’s serum (containing potential antibodies) with a known antigen and a controlled amount of complement. If specific antibodies are present in the serum, they will bind to the antigen, forming immune complexes that fix the complement.
• Secondary (Indicator) Stage: After the initial incubation, an “indicator system” is added. This system consists of sheep red blood cells (SRBCs) that have been pre-sensitized with an anti-SRBC antibody, known as haemolysin.
  • If complement was fixed in the primary stage (indicating the presence of specific antibodies), there will be no free complement left to lyse the added sensitized SRBCs. This results in no haemolysis (a positive test).
  • If no specific antibodies were present in the patient’s serum, no immune complexes formed, and the complement remains free. This free complement then lyses the added sensitized SRBCs, resulting in haemolysis (a negative test).

Explaining the Term “Complement”

Definition: The “complement system” is a vital part of the innate immune system, comprising a cascade of over 30 soluble proteins and membrane-bound receptors. These proteins, mostly synthesized by the liver, circulate in the blood as inactive precursors.

Role in Immunity: Upon activation, the complement cascade plays a crucial role in host defense mechanisms, including:

• Lysis of Pathogens: Formation of the Membrane Attack Complex (MAC), which punches holes in microbial cell membranes, leading to cell death.
• Opsonization: Coating pathogens with complement proteins (e.g., C3b), making them more susceptible to phagocytosis by immune cells.
• Inflammation: Production of anaphylatoxins (e.g., C3a, C5a) that recruit inflammatory cells and increase vascular permeability.
• Immune Complex Clearance: Removing immune complexes from circulation.

Properties Relevant to CFT:

• Lability: Complement is highly heat-labile, meaning its activity is destroyed by heating (e.g., 56°C for 30 minutes). This property is utilized in CFT to inactivate any endogenous complement activity in patient serum before testing.
• Activation Pathways: The complement system can be activated via three main pathways: the classical, alternative, and lectin pathways. In CFT, the activation primarily occurs via the classical pathway, which is initiated by the binding of C1q to an antibody-antigen complex (specifically IgM or certain IgG subclasses).
• Source for CFT: For CFT, complement is typically obtained from the serum of a healthy guinea pig. Guinea pig serum is preferred due to its consistently high and stable complement activity.

Preparation and Standardization of Complement

For the CFT to yield accurate and reproducible results, the complement must be carefully prepared and its activity precisely standardized.

Preparation:

1. Source: Obtain fresh guinea pig serum. If not used immediately, it should be aliquoted and stored at -70°C or lyophilized to preserve activity. Repeated thawing and freezing should be avoided as it significantly reduces complement activity.
2. Dilution: The complement is typically diluted in a suitable buffer, such as physiological saline (0.85% NaCl) or, more commonly, a veronal-buffered saline (VBS) or gelatin veronal-buffered saline (GVBS), which provides optimal ionic strength and pH for complement activity.

Standardization (Complement Titration): The goal of complement standardization is to determine the “Minimal Haemolytic Dose” (MHD), which is the smallest amount of complement that will cause complete lysis of a standardized amount of sensitized sheep red blood cells (SRBCs) under defined conditions. For the actual CFT, a working dilution of complement (usually 2 MHD) is used to ensure an excess of complement for the indicator system, making the test sensitive to complement consumption.

Procedure for Complement Titration:

1. Prepare Sensitized SRBCs (Indicator System): This involves combining a known concentration of SRBCs with a standardized amount of haemolysin (see Section 5 for detailed preparation).
2. Prepare Complement Dilutions:
   • Starting from a pre-diluted complement stock (e.g., 1:10 or 1:20), prepare a series of serial two-fold or four-fold dilutions in VBS/GVBS (e.g., 1:40, 1:60, 1:80, 1:100, 1:120, 1:140, 1:160).
   • Pipette a fixed volume (e.g., 0.25 mL or appropriate volume for the assay format) of each complement dilution into separate tubes or microtiter wells.
3. Add Sensitized SRBCs: Add a fixed volume (e.g., 0.25 mL) of the prepared sensitized SRBCs to each tube/well containing the complement dilutions.
4. Incubate: Incubate the tubes/wells at 37°C for 30-60 minutes.
5. Read Results: Observe for haemolysis. The MHD is the highest dilution (smallest amount) of complement that causes 100% lysis of the sensitized SRBCs.
6. Calculate Working Dilution: The working dilution of complement for the CFT typically corresponds to 2 MHD (or sometimes 2.5 MHD, depending on the specific assay protocol). For example, if 1 MHD is found at a 1:100 dilution, then 2 MHD would be at a 1:50 dilution.

Preparation and Standardization of Haemolysin

Haemolysin is an antibody (typically raised in rabbits) directed against sheep red blood cells (SRBCs). It is crucial for the indicator system, as it sensitizes the SRBCs, making them susceptible to lysis by complement.

Preparation:

1. Source: Haemolysin is usually obtained commercially as lyophilized rabbit anti-sheep red blood cell serum.
2. Reconstitution: Reconstitute the lyophilized haemolysin according to the manufacturer’s instructions, usually with sterile distilled water or physiological saline. Store aliquoted portions at -20°C or -70°C.

Standardization (Haemolysin Titration): The goal of haemolysin standardization is to determine the “Minimal Haemolytic Amount” (MHA) or “Unit,” which is the smallest amount of haemolysin that, in the presence of excess complement, will cause complete lysis of a fixed amount of SRBCs. For the CFT indicator system, a working dilution of haemolysin (typically 2 MHA) is used.

Procedure for Haemolysin Titration:

1. Prepare SRBC Suspension: Wash sheep red blood cells thoroughly with VBS/GVBS until the supernatant is clear. Prepare a 2-5% suspension (e.g., 2.5% v/v in VBS/GVBS).
2. Prepare Haemolysin Dilutions:
   • Prepare a series of serial two-fold dilutions of haemolysin (e.g., 1:100, 1:200, 1:400, 1:800, 1:1600, 1:3200) in VBS/GVBS.
   • Pipette a fixed volume (e.g., 0.25 mL) of each haemolysin dilution into separate tubes/wells.
3. Add SRBCs: Add a fixed volume (e.g., 0.25 mL) of the 2.5% SRBC suspension to each tube/well.
4. Incubate (Sensitization): Incubate the haemolysin-SRBC mixture at room temperature for 15-30 minutes to allow the haemolysin to bind to the SRBCs (sensitization).
5. Add Standardized Complement: After sensitization, add a fixed, excess amount of standardized complement (e.g., 2 MHD from the previous standardization) to each tube/well. Usually, 0.25 mL of diluted complement is added.
6. Incubate (Lysis): Incubate the tubes/wells at 37°C for 30-60 minutes.
7. Read Results: Observe for haemolysis. The MHA is the highest dilution (smallest amount) of haemolysin that causes 100% lysis of the SRBCs.
8. Calculate Working Dilution: The working dilution of haemolysin for preparing the indicator system is typically 2 MHA. For example, if 1 MHA is found at a 1:1600 dilution, then 2 MHA would be at a 1:800 dilution.

Preparation of an Indicator System (Sensitized Red Blood Cells)

The indicator system, also known as the haemolytic system, consists of sheep red blood cells (SRBCs) sensitized with haemolysin. This system is crucial for detecting the presence of free (unfixed) complement in the secondary stage of the CFT.

Procedure for Preparing Sensitized SRBCs:

1. Prepare Sheep Red Blood Cells (SRBCs):
   • Obtain fresh sheep blood (anticoagulated with Alsever’s solution or similar).
   • Wash the SRBCs by centrifugation (e.g., 800-1000 x g for 5 minutes) in a large volume of cold VBS/GVBS until the supernatant is clear and free of haemoglobin. Typically, 3-4 washes are sufficient. Decant the supernatant carefully after each wash.
   • After the final wash, aspirate the supernatant completely.
   • Prepare a precise suspension of SRBCs (e.g., 2.5% v/v) in VBS/GVBS based on your assay’s requirements. This often involves determining the packed cell volume (PCV) and diluting accordingly.
2. Sensitization of SRBCs:
   • To sensitize the SRBCs, mix an equal volume of the prepared 2.5% SRBC suspension with an equal volume of the working dilution of haemolysin (2 MHA concentration determined in Section 4).
   • Example: If you need 10 mL of sensitized SRBCs, combine 5 mL of 2.5% SRBCs with 5 mL of 2 MHA haemolysin.
   • Gently mix the suspension and allow it to stand at room temperature for 15-30 minutes. This incubation allows the haemolysin antibodies to bind to the surface of the SRBCs. The sensitized SRBCs should appear uniformly turbid, with no visible aggregates.

This prepared “sensitized SRBCs” suspension is now ready for use in the second stage of the complement fixation test. It should be used promptly as its stability is limited.

Carrying Out Complement Fixation Tests Proper

The actual execution of the CFT involves meticulous setup of test reactions and various controls to ensure validity and accurate interpretation. The test is typically performed in tubes or microtiter plates.

Components Required for the Test:

1. Test Serum (Patient Sample): Serum from the patient, previously heat-inactivated at 56°C for 30 minutes to destroy any inherent complement activity, thus preventing false positives. Serial dilutions of serum may be prepared to titrate antibody levels.
2. Known Antigen: Specific antigen related to the disease being tested. The antigen concentration must be optimized (e.g., by block titration) to ensure maximum sensitivity and specificity.
3. Standardized Complement: Working dilution (e.g., 2 MHD) prepared as described in Section 3.
4. Indicator System: Sensitized SRBCs prepared as described in Section 5.
5. Diluent: Veronal-buffered saline (VBS) or gelatin veronal-buffered saline (GVBS).

Procedure (Two Stages):

Stage 1: Primary (Complement Fixation) Stage

1. Prepare Test Reactions:
   • For each patient serum to be tested, set up at least one test well/tube. If titrating antibody, set up multiple wells/tubes for serial dilutions of the serum.
   • Test Well/Tube:
     • Add a fixed volume of known Antigen.
     • Add a fixed volume of heat-inactivated Test Serum (or its dilution).
     • Add a fixed volume of Standardized Complement (2 MHD).
     • Bring the total volume to a standard amount with diluent.
2. Set Up Controls (Crucial for Valid Results):
   • Serum Control (Anticomplementary Control): Test Serum + Complement + Diluent (NO antigen). This controls for non-specific complement consumption by the patient’s serum itself (anticomplementary activity). Haemolysis should occur (indicating no non-specific fixation). If no haemolysis, the serum is anticomplementary, and the test is invalid for that sample.
   • Antigen Control: Antigen + Complement + Diluent (NO test serum). This checks for anticomplementary activity of the antigen preparation. Haemolysis should occur.
   • Known Positive Control: Known positive antiserum + Antigen + Complement. Should show no haemolysis.
   • Known Negative Control: Known negative antiserum + Antigen + Complement. Should show haemolysis.
3. Incubation: Incubate all tubes/wells at 4°C overnight (16-18 hours) or at 37°C for 30-60 minutes. The longer 4°C incubation often increases sensitivity.

Stage 2: Secondary (Indicator) Stage

1. Add Indicator System: After the primary incubation, without disturbing the mixture, add a fixed volume of freshly prepared Sensitized SRBCs to every tube/well from Stage 1.
2. Set Up Additional Controls (Indicator System and Reagents): These are typically set up separately or alongside the main test.
   • Complement Control (Haemolytic Control): Standardized Complement + Indicator System + Diluent. This confirms the activity of the complement and indicator system. It must show 100% haemolysis. If not, the complement or indicator system is faulty, and the test is invalid.
   • Haemolysin Control: Haemolysin (working dilution) + SRBCs + Diluent (NO complement). Should show no haemolysis, confirming haemolysin alone does not lyse SRBCs.
   • Red Blood Cell Control: SRBCs + Diluent (NO haemolysin, NO complement). Should show no haemolysis, confirming SRBCs do not auto-lyse.
3. Secondary Incubation: Incubate all tubes/wells at 37°C for 30 minutes, or until the Complement Control shows complete haemolysis.

Reading and Interpretation of Results:

After the secondary incubation, centrifuge the tubes/plates lightly (optional for plates) and observe for the degree of haemolysis.

• Positive Result (Presence of Antibody/Antigen): No haemolysis or significantly reduced haemolysis. This indicates that the complement was fixed by the antigen-antibody complex in the primary stage, leaving no free complement to lyse the sensitized SRBCs. Results are typically graded from 0 (no haemolysis/complete fixation) to 4+ (complete haemolysis/no fixation). A “positive” result correlates with 0 to 25% haemolysis (3+ to 4+ fixation).
• Negative Result (Absence of Antibody/Antigen): Complete haemolysis (100% lysis). This indicates that no specific antigen-antibody reaction occurred in the primary stage, leaving the complement free to lyse the sensitized SRBCs. Correlates with 100% haemolysis (0 fixation).

Interpretation of Controls:

• Serum Control: Must show complete haemolysis. If not, the patient’s serum has anticomplementary activity, and the test result for that sample is unreliable.
• Antigen Control: Must show complete haemolysis. If not, the antigen preparation is anticomplementary, and the test is invalid.
• Complement Control: Must show complete haemolysis. If not, the complement or indicator system is inactive.
• Haemolysin Control & SRBC Control: Must show no haemolysis.

Potential Issues:

• Anticomplementary Activity: If the patient’s serum or antigen preparation non-specifically consumes complement, it can lead to false positive results. This is detected by the serum and antigen controls.
• Prozone Effect: Excessively high concentrations of antibody in the patient’s serum can sometimes inhibit antigen-antibody complex formation or complement fixation, leading to a false negative result, especially when testing undiluted serum. This is why serial dilutions are often performed.

Conclusion

The Complement Fixation Test, despite its intricate multi-component nature and relative complexity compared to modern immunoassays, remains a robust and valuable serological technique. Its success hinges on the precise standardization of each reagent and careful adherence to the procedural steps and control setup. By understanding the underlying principles and mastering the preparation and execution protocols, laboratories can reliably utilize CFT for the effective diagnosis and epidemiological surveillance of various infectious and autoimmune diseases.