By Sanjiv
Sawock
Email: rajsawock@hotmail.com
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.
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
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 -70°C 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.
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.