Complement Fixation Test (CFT)

  


1. Objective

The objective of the Complement Fixation Test (CFT) was to detect the presence of specific antibodies or antigens in a patient's serum by observing complement-mediated lysis of indicator red blood cells.

2. Principle

The test was based on the ability of antigen–antibody complexes to fix complement. If the patient's serum contained specific antibodies, they formed a complex with the added antigen and fixed the complement. When sheep red blood cells (SRBCs) coated with anti-sheep RBC antibodies were added, no hemolysis occurred, indicating a positive result. If there were no antibodies, the complement remained free and lysed the indicator SRBCs, indicating a negative result.

3. Materials Used

  • Patient serum (heat-inactivated)

  • Known antigen

  • Complement (usually guinea pig complement)

  • Sheep red blood cells (SRBCs)

  • Hemolysin (anti-sheep RBC antibodies)

  • Controls (positive and negative)

  • Incubator and water bath

4. Procedure

  1. The patient's serum was heat-inactivated to destroy native complement.

  2. Known antigen and complement were added to the serum.

  3. The mixture was incubated to allow antigen–antibody interaction and complement fixation.

  4. Sensitized SRBCs were added to the mix.

  5. After further incubation, the degree of hemolysis was observed.

5. Result Interpretation

  • Positive CFT: No hemolysis (complement was fixed by antigen–antibody complex).

  • Negative CFT: Hemolysis present (complement was free to lyse indicator RBCs).

6. Applications

  • Diagnosis of infections: e.g., syphilis, Q fever, psittacosis, echinococcosis

  • Detection of autoimmune or allergic responses

Comments

Post a Comment

Popular posts from this blog

Urine Culture

Image
  1. Objective To detect and identify pathogenic bacteria (or fungi) in urine and determine their antibiotic susceptibility , especially in suspected urinary tract infections (UTIs) . 2. Principle Urine is inoculated onto differential and selective media to isolate and identify uro-pathogens such as E. coli , Klebsiella , Proteus , and Enterococcus . A colony count is performed to assess the significance of bacterial growth, and susceptibility testing is carried out using methods like Kirby-Bauer disc diffusion . 3. Materials Sterile midstream urine sample Inoculating loop (1 µL or 10 µL calibrated) Culture media: Cystine Lactose Electrolyte Deficient (CLED) agar MacConkey agar Blood agar Incubator (35–37°C) Antibiotic discs (for sensitivity testing) Mueller-Hinton agar (for disc diffusion) PPE and sterile containers 4. Procedure Mix the urine sample gently. Use a calibrated loop to streak the urine onto culture media plate...
Read more

Coombs Test (Antiglobulin Test), including both the Direct and Indirect versions

Image
🧪 1. Objective The objective of the Coombs test was to detect antibodies (or complement proteins) bound to red blood cells (Direct Coombs) or free antibodies in a patient’s serum that could react with red cells (Indirect Coombs). It was used to diagnose immune-mediated hemolytic anemia, hemolytic disease of the fetus and newborn (HDFN), and for crossmatching in transfusions. 🧪 2. Principle The test relied on the use of Coombs reagent (anti-human globulin, AHG), which binds to human IgG and/or complement (C3d) attached to red cells. In the Direct Coombs test (DAT), it detected in vivo sensitization of RBCs. In the Indirect Coombs test (IAT), it detected antibodies in the serum capable of binding to RBC antigens in vitro. Agglutination after addition of AHG indicated a positive result. 🧪 3. Materials Patient’s red blood cells (for DAT) or serum (for IAT) Screening or panel red cells (for IAT) Anti-human globulin reagent (polyspecific or monospecific: anti-Ig...
Read more

Antiphospholipid Antibody Test (e.g., dRVVT)

Image
  1. Objective: To detect antiphospholipid antibodies in patient plasma, especially in suspected cases of antiphospholipid syndrome, using the Dilute Russell’s Viper Venom Time (dRVVT) test. 2. Principle: The dRVVT measures the time it takes for plasma to clot after the addition of diluted Russell's viper venom, which directly activates factor X. In the presence of antiphospholipid antibodies, especially lupus anticoagulant, the clotting time is prolonged. A confirmatory test with excess phospholipids is used to demonstrate correction. 3. Materials Required: Patient citrated plasma Normal pooled plasma (control) Russell’s viper venom reagent (screening and confirmatory) Calcium chloride Platelet-poor plasma Coagulometer or water bath with stopwatch Test tubes and pipettes 4. Procedure: Collected citrated blood and prepared platelet-poor plasma. Added venom reagent to both patient and control plasma. Added calcium chloride to initiate clottin...
Read more