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Measurement of Complement

 

 

By Sanjiv Sawock

 

Email: rajsawock@hotmail.com

 

Introduction

 

Measurement of complement is part of the service commitment of a clinical

immunology laboratory. There are two aspects of this service:

 

(a)                measurement of complement activation as an aid to disease diagnosis and also the assessment of disease status in many chronic diseases in which complement activation occurs.

(b)               The detection and diagnosis of inherited deficiencies complement components.

 

The main diseases in which monitoring of complement activation plays a role are the chronic inflammatory rheumatic diseases, e.g., rheumatoid arthritis and systemic lupus erythematosus and in various forms of glomerulonephritis. As complement plays a major in elimination of bacteria and immune complexes, it is not surprising that inherited complement deficiencies are associated with increased risks of developing severe recurrent bacterial infections and immune complex diseases.

 

Methods of measuring complement

Immunochemical and functional assays can be used for measuring individual components of the complement cascade. The immunochemical methods include rocket immunoelectropheresis, single radial immunodiffusion (RID), nephelometry and enzyme-linked immunoadsorbent assay (ELISA).

 

In clinical laboratory, the speed with which results are available, and the number of samples which can be assayed simultaneously are important. For the immunochemical assays, RID can handle fairly large numbers of samples, but results take between 2 and 7 days. Rocket immunoelectrophoresis is time consuming but can handle similar numbers of samples and give results in 24 hours, while nephelometry can handle about 100 samples per day on a small bench-top nephelometer. ELISA can handle up to 200 samples simultaneously and give results in a few hours but this method remains to be fully evaluated in the routine laboratory. All immunochemical assays will also measure functionally inactive protein fragments but these are cleared up so rapidly in vivo that this is not a problem in practice.

 

With functional assays, the results are available within a few hours although the number of samples that can be handled is fairly small.

 

Measurement of complement activation by immunochemical assays has relied upon measurement of the serum levels of a single component of the classical (C4), alternative (B), and the terminal sequence (C3). When taken together the CHO50 assay and immunological assays of C3, C4, and B can yield important diagnostic information (Table 1).

Pattern

Profile

Example

 

CH50

C4

C3

B

 

Classical

N

Serum sickness. SLE. RA with vasculitis. Essential mixed cryglobulinaemia. Systemic vasculitis. Hepatitis B antigenaemia

Alternative

N

Gram-negative bacteraemia. Pancreatitis. Post-streptococcal glomerulonephritis

Classical +

Alternative

SLE, shock syndromes, Type I membrano-proliferative glomerulonephritis

Fluid-phase

classical

N

N

Hereditary angio-oedema. Vivax malaria

Fluid-phase

Alternative

N

C3b inactivator or b1H defiency.

C3 nephritic factor

Acute phase

protein

Acute and chronic infections. Inflammatory disorders. Pregnency

Deficiency

N

N

N

Complement defiency other than C3, C4, or B. Collection artefact

Table 1. Patterns of complement activation

 

Interpretation of complement component levels and Normal range

 

Low levels of complement components may indicate either complement consumption or deficiency. Further tests of function and detection of activation products are needed to distinguish these antities. Functional assays such as total haemalytic complement (CH50) assay, alternative pathway haemolytic complement (AP-CH50), the terminal sequence (C3-C9) haemolytic activity as well as the haemolytic assays for individual components are used to detect complement component deficiencies.

 

Raised levels of complement components, are particularly C3 and C4, merely reflect an acute phase response. However, an acute phase response may obscure complement consumption since the rate of breakdown may be matched by the increase rate of synthesis with resultant normal levels in the serum. Assays for complement activation products are useful in this situation.

 

Determination of a normal range for the complement components is beset by several problems. There are no internationally defined standards and therefore each laboratory setting up assays must define its own range of normal values. Defining a so-called normal population is also difficult for a number of reasons:

                    Null alleles of C4A and C4B occur frequently in the normal population.

                    Serum levels of complement components tend to increase during infections as part of an acute phase response. It is therefore important to exclude individuals with inter current infections from the control population.

                    Women who are pregnant or taking oral contraception have elevated complement levels and should thus be excluded.

 

 

Handling of samples

The majority of contradictory or un-interpretable results from complement assays can be traced to improper handling or storage of specimens. As heat-labile proteins, samples for functional assays must be handled and collected under careful conditions. For some immunochemical assays this is also very important , as poor handling can lead to cleavage of C3 and C4 which results in artificially high levels for these components if assayed by RID.

 

Serum is used for CH50, AP-CH50, C3-C9 haemolytic assays and for the immunochemical assay of individual components. For assays of the activation products or anaphylatoxins, blood must be collected in 10 mM EDTA to prevent coagulation and subsequent complement activation, as this could alter the level of activation product in the sample. The plasma should be assayed directly or divided into aliquots and stored at -70C within 3 h of venepuncture. Whilst occasional freezing and thawing does not affect samples for immunochemical assays, it should always be avoided for samples to be used for functional assays.

 

Case Study: Systemic lupus erythematosus

A 24-years-old woman presented in June 2001 with fever, arthragia, myalgia, alopecia, and leucopenia. Serun IgG was elevated (20.8 g/L), C4 reduced (0.08 g/L) but C3 was normal (1.2 g/L). ANA was positive (1/1600) and DNA binding was 57% (normal <30%). A diagnosis of SLE was made. She was treated with salicylates and prednisolone and improved clinically; by October 2001 complement levels were normal but DNA binding was still elevated (47%) and normalised only in February 2002. A year later she relapsed, and was readmitted with fever, weight loss, proteinuria, and haematuria. ANA>1/1600, C3 and C4 were reduced (0.4 g/L and 0.02 g/L respectively), DNA binding was increased (96%). She was treated with high dose prednisolone and rapidly improved clinically, C3 and C4 initially and then DNA binding gradually returned to normal.

 

Serum complement levels is a reflection of the balance between synthesis and consumption. They may appear normal or be increased secondary to an acute phase response in spite of breakdown in the disease process. Samples taken at single time point may be misleading and serial determinations provide more meaningful data. A persistently low C4 does not necessarily indicate active disease.

 

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