Complement Fixation Test-Introduction, Principle, Testing Procedure, Result -Interpretation, and Keynotes

Introduction

Complement Fixation Test is a classical assay that detects specific antibodies in patient serum samples. The test employs complement proteins to reveal immune reactions between antigens and antibodies with accuracy. Moreover, the assay fixes complement when antigen-antibody complexes form quickly during incubation under controlled conditions. Consequently, any unbound complement lyses indicator red blood cells in a subsequent reaction phase efficiently.

Principle

The principle of the Complement Fixation Test relies on the classical antigen-antibody interaction mechanism with precision. Initially, antigens bind to specific antibodies to form immune complexes during the incubation period successfully. Subsequently, the formed immune complexes fix complement and rapidly inactivate free complement proteins with efficiency. Consequently, unbound complement remains available to lyse indicator cells in the assay for clear results. Additionally, the degree of cell lysis reflects the concentration of specific antibodies present quite accurately.

Testing Procedure

Initially, technicians prepare serum samples and complement reagents following strict laboratory protocols with utmost care. Subsequently, technicians mix patient serum with known antigens and add complement accordingly under controlled conditions. Then, they incubate the mixture at 37°C for one hour with gentle agitation to ensure. Afterward, technicians add sheep red blood cells as indicator cells to each reaction tube carefully. Furthermore, they incubate the tubes again to allow unbound complement to lyse red blood cells. Finally, technicians examine the tubes for hemolysis, comparing results against established positive and negative controls.

Result – Interpretation

Initially, technicians observe the extent of hemolysis in each test tube under controlled lighting carefully. If complete hemolysis occurs, the complement remains unbound and the test is negative, indicating no reaction.

Conversely, the absence of hemolysis signifies that the complement fixed by immune complexes is present and active. Subsequently, technicians compare test tubes with control tubes to confirm proper assay performance for accuracy. Moreover, positive results indicate specific antibodies in the patient’s serum have fixed complements with certainty.

Keynotes

1. The test detects specific antibodies by measuring complement fixation during antigen-antibody reactions.
2. It relies on immune complexes to consume complement proteins accurately.
3. Technicians prepare and standardize reagents under strict laboratory protocols.
4. Sheep red blood cells serve as indicator cells in the assay.
5. Free complement lyses indicator cells when it is not fixed.
6. The lack of hemolysis confirms complement fixation by antibody-antigen complexes.
7. Proper controls ensure the accuracy of each test run.
8. Timely incubation enhances test sensitivity and reliability.
9. The assay remains cost-effective and accessible in clinical settings.
10. Clinicians interpret the results to guide diagnosis and treatment decisions.

Further Readings

• https://link.springer.com/10.1007/3-540-29662-X_735
• https://bio.libretexts.org/Bookshelves/Microbiology/Microbiology_(Boundless)/12%3A_Immunology_Applications/12.02%3A_Immunoassays_for_Disease/12.2G%3A_Complement_Fixation
• https://pubmed.ncbi.nlm.nih.gov/1960542/
• https://www.mountsinai.org/health-library/tests/complement-fixation-test-to-c-burnetii
• https://sitesv2.anses.fr/en/minisite/lrue-brucellose/complement-fixation-test
• https://www.clinicalmicrobiologyandinfection.com/article/S1198-743X(14)63102-5/fulltext
• https://stacks.cdc.gov/view/cdc/55911
• https://www.simpios.eu/wp-content/uploads/2024/04/v18_3_2_enFissazioneComplemento2013.pdf
• https://taylorandfrancis.com/knowledge/Medicine_and_healthcare/Hematology/Complement_fixation_test/
• https://www.nature.com/articles/208200c0