Complement testing—past, present, and future

For more than 60 years, clinicians have been assessing complement components to measure deficiencies or abnormalities. It is very important to understand the causes of low or absent complement so that appropriate treatment and care may be given. Complement analysis is also used to define disease severity and response to therapy, making it very useful for patient management.

History of complement

How did “complement” get its name? In the late 19th century, the focus of scientific research was on the human body’s defense against microbial infection. Scientists tried to ascertain what part of the blood destroyed invading bacteria. The Theory of Metchnikoff proposed that phagocytes ingested and destroyed invading bacteria and therefore provided the basis of innate cellular immunity. Further, Paul Ehrlich provided the theoretical basis for immunology; he surmised that between the antigen and the antibody there was an additional immune molecule called a “complement.”¹ Paul Ehrlich and Ilya Ilich Metchnikoff were both awarded the Nobel Prize for Medicine in 1908.

What is the Complement System?

The Complement System is composed of 30 blood proteins that are numbered as complement proteins: C1 to C9 with some appended with additional letter designation, e.g. C3c, C5a, C5b. These proteins function in an ordered fashion to defend against infection, coat microbes, send chemical signals, and create the membrane attack complex (MAC), which results in lysis of the pathogen.

Most complement proteins are produced in the liver. There are three key biochemical pathways that activate the complement system: Classical Pathway, Alternative Pathway, and Lectin Pathway. All of these pathways have distinct recognition molecules or activation triggers.

Three reasons to measure complement

There are a several reasons why complement component measurements are requested to assess deficiencies or abnormalities in the complement system.

First, clinicians may be looking to see whether a patient has an absence of a specific protein, or a protein that is non-functional. This is generally a primary immunodeficiency. Second, the clinician could be looking for consumption, where an over-activation of complement pathways results in low levels of complement proteins. Third, a relatively new area for complement measurement is monitoring patients who are on immunosuppressive drugs.

Clinical presentations vary

The clinical presentations of patients with complement abnormalities usually fall into one of the following categories:

• Multi-system rheumatic diseases, such as systemic lupus erythematosus, juvenile rheumatoid arthritis, and Crohn’s disease
• Kidney disease
• Hemolytic anemia
• Recurrent or overwhelming infections

“The value of 50”

CH50 testing is the most common assay used to screen patients for the functional activity of the classical complement pathway, and in the work up of complement deficiency. Guidelines from The National Immune Deficiency Foundation and the European Society for Immunodeficiencies recommend screening with the CH50 assay in the diagnostic workup of complement deficiency.²

CH50 uses the Classical Pathway and its components of the pathway: C1 through C9. Each of these components is required to be activated in order obtain a normal value indicating that the immune system is effectively eliminating pathogens, damaged cells, etc. The starting point of the pathways is at the C1 complex which recognizes antibody molecules and initiates a cascade. Next is a conformational change allowing activation and cleavage of the next protein in the complement pathway. This is followed by a series of cleavage events—where one protein activates another, which activates another, then another—like a row of falling dominoes—until a protein called C5 convertase is generated. This C5 convertase cleaves complement protein 5 or C5 into C5a and C5b, initiating the formation of the MAC. The MAC is a C5b-9 complex, which can cause pores or holes to form on the surface of the pathogen or cell that the body needs to eliminate, ultimately causing its destruction. It is the formation of these membrane attack complexes that are detected and measured when 50 percent of them are lysed, thus the name “CH50.”

How to test for CH50

Presently, there are three methodologies for testing CH50:

1. Hemolytic: an old, complex, and laborious method first described by the esteemed Manfred Mayer in 1958. It uses sheep erythrocytes sensitized with anti-sheep antibodies. Complement activation leads to MAC formation and 50 percent hemolysis of the erythrocytes. Increased complement activation gives increased hemolysis, and the amount of hemoglobin release results in a red color. The more hemolysis, the darker the red color.
2. ELISA: uses a microtiter plate that combines principles of the hemolytic method; hemolysis is measured spectrophotometrically. The assay takes more than 2.5 hours to complete.
3. Liposome: a relatively new method that is an automated assay. The process entails antigen labeled liposomes sensitized by antibodies. The complements are activated by the antigen antibody complex that will break the liposome membrane. An enzyme called Glucose 6-phosphtase converts nicotinamide adenine dinucleotide (NAD) to NADH (nicotinamide adenine dinucleotide (NAD) + hydrogen (H)), and the rate of production of NADH is then measured. Test results are available in 13 to 15 minutes on specific automated analyzers.

Because of the different testing methods for the CH50 assay, there will be variances in what is being measured and in the type of units a laboratory will be using. The numerical values across the different methods may not be directly comparable. Causes for the differences in results include the detection technologies, assay optimization protocols, different cutoffs for normal ranges, and specific laboratory conditions.

Integrity is key

Sample integrity is the single most important factor when running the CH50 assay. Since CH50 complement is labile, preserving sample integrity is critical for accurate results. CH50 activity gradually decreases with time and heat. Samples should be frozen, kept on ice, or run immediately.

Future of complement testing

The future is very promising. Great progress has been made in understanding of the quantification and activation of the complement system. Recently, complement has been associated with neurodegenerative disorders, such as Alzheimer’s disease, multiple sclerosis and Guillain-Barre syndrome.^3,4 Other complement assays that clinicians may order to look at complement disease associations or deficiencies may include:
C1 inactivator, C1q, C2, C3c, C4, C5-9, Factors B, H, and I. Scientists in complement research are continuing to harness the complement system for solutions in the understanding of how complement measurements work in diagnosis of diseases, drug therapies, and solutions for immunodeficiencies.

REFERENCES

1. Gordon S., J. Immunol. 2008. Sir Wm. Dunn School of Pathology, University of Oxford, Oxford, UK. 38:3257-3264.
2. Immune Deficiency Foundation [IDF] Diagnostic Care and Clinical Care Guidelines. Diagnostic & Clinical Care Guidelines for Primary Immunodeficiency Diseases, 3^rd Edition 2015.
3. Kirschfink M, Mollnes T. Modern Complement Analysis. Clinical and Diagnostic Laboratory Immunology, Nov. 2003. P. 982-989.
4. Mukherjee P, G.M. Pasinetti. 2000. The role of complement anaphylatoxin C5a in neurodgeneration: implications in Alzheimer’s disease. Neuroimmunology 105: 124-130.