Methods for Quantitative Detection of Antibody-induced Complement Activation on Red Blood Cells

Summary

Here we describe two assays for measuring complement activation induced by antibodies against red blood cells. The major advantage over the current assays is their quantitative and easy-to-interpret nature.

Abstract

Antibodies against red blood cells (RBCs) can lead to complement activation resulting in an accelerated clearance via complement receptors in the liver (extravascular hemolysis) or leading to intravascular lysis of RBCs. Alloantibodies (e.g. ABO) or autoantibodies to RBC antigens (as seen in autoimmune hemolytic anemia, AIHA) leading to complement activation are potentially harmful and can be - especially when leading to intravascular lysis - fatal¹. Currently, complement activation due to (auto)-antibodies on RBCs is assessed in vitro by using the Coombs test reflecting complement deposition on RBC or by a nonquantitative hemolytic assay reflecting RBC lysis^1-4. However, to assess the efficacy of complement inhibitors, it is mandatory to have quantitative techniques. Here we describe two such techniques. First, an assay to detect C3 and C4 deposition on red blood cells that is induced by antibodies in patient serum is presented. For this, FACS analysis is used with fluorescently labeled anti-C3 or anti-C4 antibodies. Next, a quantitative hemolytic assay is described. In this assay, complement-mediated hemolysis induced by patient serum is measured making use of spectrophotometric detection of the released hemoglobin. Both of these assays are very reproducible and quantitative, facilitating studies of antibody-induced complement activation.

Introduction

Antibodies against red blood cells (RBCs) can be induced by transfusion of RBCs expressing an antigen which is not present on recipient RBCs. These allo-antibodies can cause severe acute hemolytic transfusion reactions due to complement activation upon the following transfusion⁵. In auto-immune hemolytic anemia (AIHA), patients have auto-antibodies against their own RBCs. This leads to accelerated clearance of the cells via the interaction of IgG bound to RBCs with Fcγ-receptors on phagocytes in the spleen and/or in case of auto-antibodies able to activate complement via complement receptors in the liver^6,7. Fulminant complement activation r....

Protocol

1. Preparation of Bromelain-treated Red Blood Cells

1. Wash 0-typed red blood cells 3x with 1x PBS (centrifugation at 664 x g for 2-5 min).
2. Incubate 1 volume of packed RBCs with two volumes of a 0.5% bromelain solution for 10 min at 37 °C.
3. Wash the cells 3x with 1x PBS.
4. The cells can be stored as a 3% solution at 4 °C in 1x PBS for at least one week.

2. C3 and C4 Deposition Analysis on RBCs by FACS

1. Heat-inactivate the required amount of patient serum (or other anti-RBC antibody source) by heating the sample for 30 min at 56 °C.
2. Wash bromelain-treated RBCs thr....

Results

Figure 2A shows a representative scatter plot for RBCs. Suitable gating for single RBCs can be seen in de FSC-W - FSC-A plot (P1). Usually around 95% of the RBCs fall within this P1 gate (single cells), but when a high percentage of patient serum is used, this can drop to 70-80%, especially when anti-RBC IgM is present in the patient serum.

Representative results for the effect of AIHA patient serum on C4 deposition are shown in Figure 2B and on C3 deposition .......

Discussion

The above assays are reproducible and robust. They are easy to perform and it is possible to perform them with many samples at the same time (in a 96-well plate). Hence, this approach would also be suitable for a fully automated system, e.g. an ELISA robot system. In contrast to the currently used techniques, these assays are quantitative and this will help e.g. in the study of the effect of complement inhibitors. Moreover, the interpretation is objective, which is an improvement over the currently used.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
PBS	Fresenius Kabi
BSA	Sigma	B-4287
Barbital	Fagron	0261	This chemical is subject to drug regulation.
Sodium Barbital	Fagron	0263	This chemical is subject to drug regulation.
Gelatin	Merck	1.0470.0500
Sodium chloride	Merck	1.06404.1000
Magnesium Chloride	Merck	1.05833.0250
Calcium chloride	Merck	1.0238.0500
Dylight 488 amine reactive dye	Pierce	46402
Dylight 650 amine reactive dye	Pierce	62265
αC5 (Eculizumab)	Alexion Pharmaceuticals
FACS (Canto)	BD		Any FACS can be used that has the appropiate lasers.
Spectrophotometer (e.g. Multiskan spectrum)	Thermo Labsystems	1500-193	Any spectrophotometer with the right wavelength range can be used
BD FACSDiva software v 6.1.2	BD	643629	Any compatible FACS analysis software can be used
Bromelain	Sanquin	K1121