1 Department of Laboratory Medicine, Madre Giuseppina Vannini Hospital, Rome, Italy.
2 Department of Laboratory Medicine, Catholic University of the Sacred Heart, Rome, Italy.
3 Institute of Internal Medicine, Catholic University of the Sacred Heart, Rome, Italy.
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Cryoglobulins are immunoglobulins that precipitate in serum at temperatures below 37°C and resolubilize upon warming. The clinical syndrome of cryoglobulinemia usually includes purpura, weakness, and arthralgia, but the underlying disease may also contribute other symptoms. Blood samples for cryoglobulin are collected, transported, clotted and spun at 37°C, before the precipitate is allowed to form when serum is stored at 4°C in a Wintrobe tube for at least seven days. The most critical and confounding factor affecting the cryoglobulin test is when the preanalytical phase is not fully completed at 37°C. The easiest way to quantify cryoglobulins is the cryocrit estimate. However, this approach has low accuracy and sensitivity. Furthermore, the precipitate should be resolubilized by warming to confirm that it is truly formed of cryoglobulins. The characterization of cryoglobulins requires the precipitate is several times washed, before performing immunofixation, a technique by which cryoglobulins can be classified depending on the characteristics of the detected immunoglobulins. These features imply a pathogenic role of these molecules which are consequently associated with a wide range of symptoms and manifestations. According to the Brouet classification, Cryoglobulins are grouped into three types by the immunochemical properties of immunoglobulins in the cryoprecipitate. The aim of this paper is to review the major aspects of cryoglobulinemia and the laboratory techniques used to detect and characterize cryoglobulins, taking into consideration the presence and consequences of cryoglobulinemia in Hepatitis C Virus (HCV) infection.
Easy Definition and Classification
|Table 1. Brouet reclassification.|
Molecular Basis of Cryoprecipitation
Laboratory Testing for the Detection and Typing of CGs
|Figure 1. Brouet reclassification.|
Other Quantification MethodsTotal protein quantification is a method alternative to CRT measurement, although it is still awaiting validation. It permits evaluation of CGs concentration, although it is strongly affected by the presence of other proteins contained in cryoprecipitates such as albumin, fibronectin, C1q and other complement factors. Total protein quantification requires accurate washing of cryoprecipitates as well as complete re-suspension of CGs. It offers the advantage of greater sensitivity as opposed to CRT since it evaluates cryoprecipitates that adhere to the bottom of Wintrobe tubes and may, therefore, escape visual inspection. Musset et al. quantify total proteins in cryoprecipitates by spectrophotometric analysis at 280nm following CGs solubilization in 0.1nmol/L NaOH. Brouet et al. re-suspend CGs in 0.1mol/L of acetic acid and perform a colorimetric quantification of cryoprecipitate content of total proteins using either Pyrogallol Red or Coomassie Blue staining: 1mL of serum is stored at 4°C for 3 days and subsequently centrifuged at 5000 rpm for 5 min at 4°C. CGs are separated from supernatant serum, washed three times with 3mL of cold water and re-dissolved physiological solution at 37°C. Nephelometric quantification of albumin may detect contamination from residual serum proteins. Literature reports indicate that the reference serum cryoprecipitate total protein content values should be <20 mg/L. Other experimental quantification data may be obtained by calculating the difference between the nephelometric measurement of the total serum immunoglobulin concentration at 37°C and supernatant immunoglobulin concentration at room temperature following precipitation. An electrophoretic run of re-solubilized cryoprecipitate performed at 37°C, either using capillary electrophoresis or by agarose gel electrophoresis, provides accurate CGs quantification. It is achieved by calculating the area under the curve in the gamma region of the electropherogram profile and by subtracting the equivalent amount of co-precipitating serum globulins from this value on the basis of the amount of residual albumin. The latter is therefore used as an internal standard correction factor for cryoprecipitate measurement, by performing the following calculations: γ-globulin/albumin ratio of cryoprecipitate versus γ-globulin/albumin ratio of native serum.
Cryoglobulinemia and HCVCryoglobulinemia is considered to be a rare disorder, but its occurrence is strongly linked to the prevalence of HCV infection in the general population. Other viral infections, as Hepatitis B Virus, Epstein Barr Virus , HIV can induce, even if with but with minor frequency, mixed crioglobulinema, that is almost always type III.[9,18,47]
ConclusionThe possibility of detecting even very limited amounts of CGs may offer an invaluable resource to clinicians operating in this field. There is also a growing demand for more efficient and rapid tests for detection of their presence. Since even limited amounts of cryoglobulins may be both pathogenic and significant in certain clinical contexts, their detection at low levels may be critical for diagnosis and especially for those patients requiring plasmapheresis. A high prevalence of cryoglobulin ≤0.05 g/L in clinical practice may be responsible for severe renal and neurological complications, leading to high morbidity and mortality in these patients. Therefore both appropriate therapy and careful follow-up is required to improve such patients' outcome.[58,61]