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Methods for Quantitative Detection of Antibody-induced Complement Activation on Red Blood Cells
Published on: January 29, 2014
STUDIES ON IMMUNE HUMAN HEMOLYSIS : II. THE DONATH-LANDSTEINER REACTION AS A MODEL SYSTEM FOR STUDYING THE MECHANISM
C F Hinz1, M E Picken, I H Lepow
1Department of Medicine, School of Medicine and the Institute of Pathology, Western Reserve University, Cleveland.
Insights
Complement component C'1 is essential for the cold phase of the Donath-Landsteiner reaction, while other complement components are needed for the warm phase. This study establishes a human model for investigating complement function in disease.
Area of Science:
- Immunology
- Complement System
- Hemolytic Reactions
Background:
- The Donath-Landsteiner reaction is a crucial diagnostic tool involving complement.
- Understanding the specific roles of complement components in this reaction is vital for disease research.
Purpose of the Study:
- To elucidate the sequential role of complement components in the Donath-Landsteiner reaction.
- To establish a human model for studying complement's role in disease.
Main Methods:
- Investigating the effects of inactivating specific complement components (C'1, C'2, C'3, C'4).
- Utilizing partially purified C'1 or C'1 esterase.
- Employing a serum inhibitor of C'1 esterase.
- Eluting C'1 esterase activity from erythrocytes using Na(3)EDTA.
Main Results:
- C'1 inactivation blocks the cold phase; other component inactivation does not.
- C'1, C'4, C'2, and C'3 are sequentially required for hemolysis.
- C'4 reacts in cold or warm phases, while C'2 and C'3 require the warm phase.
- C'1 esterase activity is crucial and can be eluted.
- Calcium (Ca++) and Magnesium (Mg++) ions are required for specific steps.
Conclusions:
- C'1 esterase plays a functional role in a human disease state.
- A human model is established for studying complement mechanism of action.
Abstract:
1. The inactivation, of C'1 blocks the completion of the cold phase of the Donath-Landsteiner reaction; inactivation of the other components of complement does not interfere with the cold phase of the reaction. 2. C'2, C'3, and C'4 are required for the completion of the hemolytic reaction. C'4 reacts in either the cold or warm phase, but C'2 and C'3 must react in the warm phase. 3. Partially purified C'1 or C'1 esterase can be substituted for whole serum in the cold phase, although neither reagent contains any of the other components of complement 4. Partially purified serum inhibitor of C'1 esterase blocks the effect of C'1 esterase in the cold phase, and reverses the effect of complement, C'1 or C'1 esterase when incubated with "activated" cells after the cold phase. 5. C'1 esterase activity can be eluted from "activated" erythrocytes with Na(3)EDTA. 6. The components of human complement in this human hemolytic reaction act in the order C'1, C'4, C'2, C'3. Ca(++) is required for the reaction with C'1, and Mg(++) is required for the reaction with C'2. 7. Accordingly, a functional role of C'1 esterase in a human disease state is demonstrated, and a human model is established for the study of the mechanism of action of complement.
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