Nephelometry has become increasingly more popular in diagnostic laboratories and depends on the light-scattering proper ties of antigen-antibody complexes (Fig. 1).

Fig1. Principle of nephelometry for the measurement of antigen-antibody reactions. Light rays are collected in a focusing lens and can  ultimately be related to the antigen or antibody concentration in a sample.

measured photometrically. When specific antigen-coated latex particles acting as reaction intensifiers are agglutinated by their corresponding antibody, the increased light scatter of a solution can be measured by nephelometry as the macromolecular complex form. The use of polyethylene glycol (PEG) enhances and stabilizes the precipitates, thus increasing the speed and sensitivity of the technique by controlling the particle size for optimal light angle deflection. The kinetics of this change can be determined when the photometric results are analyzed by computer.

In immunology, nephelometry is used to measure complement components, immune complexes, and the presence of a variety of antibodies (Box 1).

Box1. Immunologic Assays Performed by Nephelometry

Principle

Formation of a macromolecular complex is a fundamental prerequisite for nephelometric protein quantitation. The procedure is based on the reaction between the protein being assayed and a specific antiserum. Protein in a patient’s specimen reacts with specific nephelometric antiserum to human proteins and forms insoluble complexes. When light is passed through such a suspension, the resulting complexes of insoluble precipitants scatter incident light in solutions. The scattered light can be detected with a photodiode. The amount of scattered light is proportional to the number of insoluble complexes and can be quantitated by comparing the unknown patient values with standards of known protein concentration.

The relationship between the quantity of antigen and measuring signal at a constant antibody concentration is expressed by the Heidelberger curve. If antibodies are present to excess, a proportional relationship exists between the antigen and resulting signal. If the antigen overwhelms the quantity of antibody, the measured signal drops.

By optimizing the reaction conditions, the typical antigen antibody reactions as characterized by the Heidelberger curve are effectively shifted in the direction of high concentration. This ensures that these high concentrations will be measured on the ascending portion of the curve. At concentrations higher than the reference curve, the instrument will transmit an out of range warning.

Physical Basis

 Nephelometry is based on the principle that light is scattered by a homogeneous particulate solution at a variety of angles. Three types of scatter can occur: (1) scatter around the particles; (2) forward scatter caused by out of phase backscatter; and (3) forward scatter exceeding backscatter.

Optical System

In the nephelometric method, an infrared high-performance, light-emitting diode (LED) is used as the light source. Because an entire solid angle is measured after convergence of this light through a lens system, an intense measuring signal is available when the primary beam is blocked off. In connection with the lens system, this produces a light beam of high colinearity. The wavelength is 840 nm. Light scattered in the forward direction in a solid angle to the primary beam ranges between 13 and 24 feet and is measured by a silicon photodiode with an integrated amplifier. The electrical signals generated are digitized, compared with reference curves, and converted to protein concentrations.

Measuring Methods

A fixed-time method is used routinely for precipitation reactions. Ten seconds after all reaction components have been mixed, a cuvette reading (initial blank measurement) is taken. A second measurement is taken 6 minutes later and, after sub traction of the original 1-second blanking value, a final answer is calculated against the multiple-point or single-point calibration in the computerized program memory for the assay.

Advantages and Disadvantages

Nephelometry represents an automated system that is rapid, reproducible, relatively simple to operate, and common in higher volume laboratories. It has many applications in the immunology laboratory. Currently, instruments using a rate method and fixed-time approach are commercially available with tests for immunoglobulin G (IgG), IgA, IgM, C3, C4, properdin, C-reactive protein (CRP), rheumatoid factor, ceruloplasmin, α1-antitrypsin, apolipoproteins, and haptoglobins.

The disadvantages of nephelometry include high initial equipment cost and interfering substances such as microbial contamination, which may cause protein denaturation and erroneous test results. Intrinsic specimen turbidity or lipemia may exceed the preset limits. In these cases, a clearing agent may be needed before an accurate assay can be performed. In addition, low-molecular-weight immunoglobulins, monoclonal immunoglobulins, and antibovine antibodies also may produce spurious results in nephelometry.

Clinical Application: Cryoglobulins

Cryoglobulin analysis is frequently requested when patient symptoms such as pain, cyanosis, Raynaud’s phenomenon, and skin ulceration on exposure to cold temperatures are present. Cryoglobulins are proteins that precipitate or gel when cooled to 0° C (32° F) and dissolve when heated. In most cases, mono clonal cryoglobulins are IgM or IgG. Occasionally, the macro globulin is both cryoprecipitable and capable of cold-induced anti-i–mediated agglutination of red blood cells.

Cryoglobulins with a detected monoclonal protein component normally prompt a clinical investigation to determine whether an underlying disease exists. Cryoglobulins are classified as follows:

 • Type I—cryoprecipitate is a monoclonal IgG, IgA, or IgM.

 • Type II—cryoprecipitate is mixed, containing two classes of immunoglobulins, at least one of which is monoclonal.

 • Type III—cryoprecipitate is mixed and no monoclonal protein is found.

To test for the presence of cryoglobulins, blood is collected, placed in warm water, and centrifuged at room temperature. The serum is then put into a graduated centrifuge tube and placed in a 4° C (39° F) environment for 7 days. If a gel or precipitate is observed, the tube is centrifuged and the precipitate is washed at 4° C (39° F), redissolved at 37° C (98.6° F), and evaluated by double diffusion and immunoelectrophoresis for the content of the cryoglobulin. Newer methods use nephelometry with cold treatment for analysis.

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