Table 19.1 Examples* of major polyclonal elevations in immunoglobulin levels

*But not sufficiently specific to be of diagnostic value in individual patients.

Serum

It is essential that sera sent for immunoglobulin quantification is screened by serum protein electrophoresis for the presence of paraproteins (monoclonal bands), since these abnormal immunoglobulins may react with the standard antisera in a different way due to variable epitopes and structural changes. The principle of the methodology is given here though most of these assays are now automated.

In protein electrophoresis, a wet membrane or gel is stretched across an electrophoresis tank, and filter-paper wicks provide a continuous buffer phase. Serum samples are applied to the surface on the cathodal side and an electric current is passed through the membrane or gel for about 45 min. It is then removed and the protein bands visualized with an appropriate dye (Fig. 19.3). A normal serum is always run with the test specimens for comparison and quality control. Since this is done by machine now, the electrophoretic strip can be visualized on the computer or printed out. Discrete monoclonal immunoglobulin (M) bands can appear anywhere along the strip and must be investigated further. False-positive bands, which are not due to immunoglobulin, may be caused by haemoglobin (in a haemolysed sample), fibrinogen (in plasma or an incompletely clotted specimen) or aggregated IgG (in a stored specimen). It is therefore important to send fresh clotted blood specimens for this test.

Fig. 19.3 Principle of serum protein electrophoresis. At the pH of routine electrophoresis (pH 8.6), serum proteins carry a net negative charge and migrate towards the anode. Some weakly charged proteins, such as immunoglobulins, are carried back towards the cathode by the flow of buffer.

When an M band is found on electrophoresis, the nature of the band must be determined by immunofixation, also automated. Several samples of the patient’s serum are first electrophoresed on agar gel (Fig. 19.4). Specific antisera to IgG, IgA, IgM and κ and λ light chains are then applied to the electrophoresed samples, by soaking strips of cellulose acetate membrane in the individual antisera and laying these strips on the surface of the gel. Precipitation (i.e. immunofixation) of the M protein is achieved by incubating the gel and antisera. Unfixed (non-precipitated) protein is washed from the gel and the ‘fixed’ bands are stained and visualized. In the absence of a heavy-chain abnormality, an abnormal reaction with antisera specific for light chains alone suggests that the M band is due to free monoclonal light chains or, very rarely, an IgD or IgE paraprotein. An abnormal reaction with a heavy-chain antiserum alone is very unusual. Individual M bands can be quantified by densitometry – also automated; this measures the intensity of stain taken up by each band and produces a tracing corresponding to the electrophoretic strip (Fig. 19.5). The proportion attributable to the monoclonal protein is expressed as a percentage of the whole tracing and converted to absolute terms (g/l) by reference to the total serum protein concentration. Scanning densitometry is the most reliable method for measuring paraprotein concentration, particularly for serial monitoring or in samples containing large amounts of non-paraprotein immunoglobulins. Quantitation by comparison of the levels of free κ and λ light chains provides another, faster way to monitor changes in paraprotein levels for clinical management in lymphoproliferative diseases (Chapter 6).

Fig. 19.4 Typing an M band by immunofixation. In this schematic example (a), the M band found on electrophoresis (i) is identified as an IgA (type λ) as shown on the actual fixation gel (b).

Fig. 19.5 Densitometric analysis of protein electrophoresis for quantification of an M band. A, Albumin. Normal trace on left.

Gross elevations of the immunoglobulin levels indicate the need to measure the relative serum viscosity (RSV); this is the time taken for a given volume of serum to pass through a narrow capillary tube, relative to water. RSV is normally 1.4–1.8. Symptoms of hyperviscosity (see Case 6.9) usually develop when the RSV value exceeds 4.0.

Provided the serum is fresh, a heavy deposit of protein at the origin in serum electrophoresis may indicate the presence of cryoglobulins. Cryoglobulins are immunoglobulins that form precipitates, gels or even crystals in the cold. The severity of the symptoms (see Case 11.7 and section 11.6.3) depends on the cryoprotein concentration and the temperature at which cryoprecipitation occurs (thermal range). If cryoglobulins are suspected clinically, a fresh specimen of blood must be taken directly into a warmed container (37°C) and delivered warm (37°C) to the laboratory; it is allowed to clot at 37°C and also separated at 37°C. Aliquots of separated serum are kept at 4°C for 24 h or longer. Centrifugation and washing of any resultant precipitate must be done at 4°C. The precipitate is redissolved by warming back to 37°C and then analysed for its constituent proteins by immunofixation at 37°C in order to determine the type of cryoglobulin (Chapter 11).

Urine

Analysis of urine is essential in suspected myeloma, any condition in which a serum M band has been found, hypogammaglobulinaemia of unknown cause and in amyloidosis.

Normal immunoglobulin synthesis is accompanied normally by production of excessive amounts of free polyclonal light chains (see section 6.6). These are excreted into the urine, where they can be detected in minute amounts in everyone if a really sensitive assay is used. Patients with renal damage excrete larger quantities of polyclonal free light chains in the urine.

Free monoclonal light chains (Bence Jones proteins) cannot be detected by routine measurement of total urinary protein or urine dipstick testing. The only reliable test for suspected ‘Bence-Jones proteinuria’ consists of three stages:

• concentration of urine;
  
• electrophoresis to demonstrate the presence of an M band; and
  
• immunofixation to confirm that the monoclonal band is composed of either monoclonal κ or λ free light chains. The excretion of a whole paraprotein by a damaged kidney may give a false-positive result, unless the free light-chain nature of the M band is confirmed or the serum paraprotein is run alongside for identification.

Cerebrospinal fluid

IgG and albumin concentrations in cerebrospinal fluid (CSF) can be measured. Since albumin is not synthesized in the brain, the relationship between IgG and albumin – the CSF IgG index – gives an indirect indication of how much CSF IgG has been synthesized by lymphocytes inside the brain (Fig. 19.6). The CSF IgG Index is given by dividing (IgG CSF/IgG serum) by (Alb CSF/Alb serum) and should be less than 0.72. The index therefore corrects for reductions in the albumin–IgG ratio associated with diseases that alter the permeability of the blood-brain barrier. In contrast to serum, the IgG in CSF is often of a restricted nature and forms oligoclonal bands, i.e. there are two or more discrete bands rather than a diffuse increase. Oligoclonal bands cannot be detected by routine electrophoresis of unconcentrated CSF and the degree of concentration needed (80-fold) to make bands visible induces artefacts. The most satisfactory method is isoelectric focusing and immunofixation with an enzyme-labelled antiserum to IgG (Fig. 19.7). This is an essential test in the investigation of demyelinating disorders such as multiple sclerosis (see Case 17.2); each set of bands is unique.

Fig. 19.6 Cerebrospinal fluid (CSF) IgG index. The y axis shows increasing values for the quotient of IgG in CSF: IgG in serum, and the x axis shows increasing values for the quotient of albumin in CSF: albumin in serum. The four areas signify: (1) normal; (2) local synthesis (normal barrier function); (3) local synthesis plus abnormal barrier function; (4) barrier function abnormal (not local synthesis).

Fig. 19.7 Oligoclonal bands of IgG detected in cerebrospinal fluid (CSF). Isoelectric focusing separates proteins within a pH gradient according to their acidic or basic nature. The proteins are then transferred to a nitrocellulose membrane by blotting and the nitrocellulose immunofixed with an antiserum to IgG to show the IgG-specific bands. The pattern is interpreted by comparing paired samples of CSF and serum (S). A positive test is one where oligoclonal IgG bands are found in CSF but not in serum.

Table 19.2 Interpretation of complement changes in disease