Detection of cryoglobulins — To detect cryoglobulin, 10 to 20 mL of blood are drawn into syringes and/or collection tubes that have been prewarmed to 37ºC without anticoagulants. These precautions are required because failure to prewarm may lead to false-negative results, due to loss of the cryoglobulin in the clotted blood (eg, if there is cooling below 37°C during collection, clotting, or centrifugation) and because the presence of anticoagulants may produce false-positive results due to the formation of cryofibrinogen or heparin-precipitable complexes.
After clotting at 37°C for one-half to one hour, the serum is separated by centrifugation at 37°C, placed in a graduated (Wintrobe) tube, and refrigerated (4°C) to allow the precipitation of cryoglobulin. In type I cryoglobulinemia, precipitates are often seen within 24 hours (sometimes in less than 90 minutes). However, three to five days are usually allowed for complete precipitation, especially for the mixed cryoglobulins, and some type II and type III cryoglobulins require up to seven days for precipitation . Most laboratories will determine a cryocrit, which is a measure of the packed (centrifuged) volume of the precipitate as a percentage of the original serum volume at 4°C.
Further confidence that the precipitate is a true cryoglobulin is obtained by washing the precipitate three to six times in cold saline solution to reduce the possibility of precipitated salts or other proteins. In addition, the precipitate can then be redissolved in saline at 37°C to confirm the warm solubility of the cryoglobulins. At this time, cryoglobulin protein concentration can be determined by spectrophotometry. Further characterization can be accomplished by immunofixation, enzyme-linked immunosorbent assay (ELISA), or another specific immunologic assay.
Some laboratories perform further testing consisting of a measurement of absolute cryoglobulin concentration, along with a description of the components of the immune complexes, including mono- or polyclonality of IgM, IgG, IgA, IgE, kappa, and/or lambda light chains. In type II cryoglobulinemia, the monoclonal component is typically IgM kappa with rheumatoid factor (RF) activity.
The cryocrit in individuals without cryoglobulinemia is close to zero; generally, a cryocrit over 0.5 to 1 percent or cryoglobulin concentration over 50 mcg/mL is considered clinically significant . The cryocrit in affected patients may approach 50 percent or may encompass the entire serum volume in type I cryoglobulinemia under conditions in which the monoclonal protein forms a gel.
The cryocrit is generally between 2 and 7 percent in type II and between 1 and 3 percent in type III disease, but there is a poor correlation between the cryocrit and clinical symptoms and features.
When cryoglobulinemia is suspected clinically, a negative result from routine laboratory testing for cryoglobulins does not exclude cryoglobulin-mediated disease . The clinician may need to draw a new specimen after consulting with the laboratory staff or clinical pathologist to assure that procedures are in place for the appropriate handling of the patient's blood when the sample is obtained and transported and to be certain that the laboratory has the necessary equipment (particularly a temperature-controlled centrifuge) to prevent premature cooling of the sample.
Bhopalwala. H
