Final Diagnosis -- Type II mixed cryoglobulinemia

FINAL DIAGNOSIS    Type II mixed cryoglobulinemia secondary to chronic HCV infection, Membranoproliferative glomerulonephritis secondary to cryoglobulinemia.


What are cryoglobulins: Cryoglobulins are immunoglobulins that precipitate as serum is cooled below core body temperature.1 Precipitation may occur just below 37C or not until the serum is cooled to near 4C. Some cryoglobulins precipitate within a few hours and others may take 2-3 days before they precipitate at 4C, therefore, cryoglobulin determination is made atleast 72 hours after the serum is maintained at 4C.

Indications of testing: Cryoglobulin testing should be carried out if the patient has clinical or laboratory features suggestive of presence of cryoglobulins (Severe cold-dependent symptomatology, Raynaud's syndrome, acrocyanosis of the digits or helices of the ears, gangrene in the absence of other known causes of vascular occlusion, livedoid vasculitis, gelling of blood on blood drawing, pathologic evidence of occlusive vasculopathy due to immunoglobulins, syndrome of cutaneous vasculitis-purpura/leg ulcers, arthralgias/arthritis, Immune-complex nephritis, Neuropathy, renal disease characterized pathologically by membranoproliferative glomerulonephritis, hyaline inclusions in glomeruli, vasculitis, chronic hepatitis with extrahepatic clinical manifestations, high-titer rheumatoid factor activity in the absence of clear-cut rheumatic disease, strikingly depressed levels of C4, Extra-articular manifestations of rheumatoid arthritis, Chronic inflammatory liver diseases, chronic inflammatory bowel diseases) or is known to have a disease that may be associated with cryoglobulins (Multiple myeloma, Waldenstrom's macroglobulinemia, chronic hepatitis C virus infection, Sjogren's syndrome, Chronic lymphocytic leukemia, Non-Hodgkins' lymphoma, cold agglutinin disease, chronic infections, autoimmune diseases, Systemic lupus erythematosus, Rheumatoid arthritis, Inflammatory bowel diseases, Biliary cirrhosis). 2

Processing of the sample: 10- 20 ml of sample should be collected into a red top tube or syringe prewarmed to 37C. Sample should be allowed to clot at this temperature for about 30- 60 mins. Serum is separated by warm centrifugation for 10 mins at 2500 rpm. Alternatively, the sample can be collected in a tube containing EDTA (yellow top tube). 2 Following separation the plasma/ serum should be checked for lipemia. It is then kept at 4C and inspected for cryoprecipitation daily for 7 days (3-7 days). Freeze-thaw of the specimen should be avoided at any point during processing, as it affects immunoglobulin solubility. The specimen can be rewarmed to 37C to check for resolubility. Some laboratories keep a parallel aliquot at 37C for comparison, and a third aliquot at 4C to be used in case of a positive result for rigorous washing (a minimum of 3 to 6 washes is required with phosphate buffered saline), resolubilization by warming, and repeat cryoprecipitation prior to detailed characterization.2

Methods of detection: The concentration of a cryoglobulin can be measured as a cryocrit. Although this is a convenient, rapid and an inexpensive method it is not standardized as to volume, tube size, or conditions for centrifugation, and should not be considered an index of disease activity when comparing different patients.2 Rapid screening and quantification of the immunoglobulins include turbidometry and nephelometry of the washed and resolubilized precipitate. Some studies have tried to determine a normal levels of cryoglobulins to be below 20 g/ml. 3


Immunofixation-Immunofixation is currently the method of choice for the typing of cryoglobulins into type I, II and III. 4 It is fast, easy to interpret, sensitivity and detects small amounts of monoclonal proteins in the serum, even in the face of a normal serum protein electrophoresis. Type I cryoglobulins: These represent monoclonal immunoglobulins an example of which is shown below in Fig 11, often present at concentrations exceeding 5 mg/ mL. 2

Type II cryoglobulins are mixed polyclonal and monoclonal immunoglobulins, frequently found in excess of 1 mg/mL. They may begin to precipitate within hours of refrigeration and are usually apparent by the next day. Our case is an example of this type of cryoglobulinemia. 2

Type III Cryoglobulins are polyclonal an example of which is shown in figure 12, and are found at levels less than 1 mg/mL. They may require several days to precipitate and may be barely visible at the base of the centrifuge tube. 2

Other tests helpful in completing the picture of cryoglobulinemia include serum viscosity, complement levels (C3, C4 and total hemolytic complement CH50) and rheumatoid factor.2


  1. The most important variable in cryoglobulin testing is maintaining the temperature at 37C while drawing the sample, its transportation and separation of serum from other blood elements.2
  2. As much as a third of the cryoprecipitate may be of non- immunoglobulin in origin example cold-precipitable complexes of fibrin and fibrinogen, C1q.2
  3. Presence of heparin in the sample leads to complex formation with fibronectin and cryoprecipitation.2
  4. Presence of fibrinogen may be mistaken for a monoclonal protein on protein electrophoresis. In this situation, thrombin may be added to the warm sample to induce clotting. 2
  5. A cryoglobulin screen may be falsely positive, and immunochemical characterization of any cryoprecipitate obtained is necessary to confirm the presence of immunoglobulin.1, 5
  6. Criteria for detection and quantitation include the removal of nonspecifically adherent serum proteins from the precipitate by serial washing which may lead to significant loss of the cryoprecipitate.

Serial studies in cryoglobulinemia: There may be considerable changes in the composition of mixed cryocomplexes over time as IgM levels fluctuate with activity of disease, decrease with treatment, or if patients become hypogammaglobulinemic.6 Thus, there is a poor correlation between laboratory parameters, such as cryoglobulin level, antiglobulin titer, or depressed C4 level, and clinical symptomatology when different patients are compared.

Association of HCV, cryoglobulinemia and non hodgkin's lymphoma: Apart from the hepatocytes, HCV infects the cells of immune system. This helps to explain not only its persistence due to ability to escape the immune response but also the development of immunologic abnormalities including cryoglobulinemia and frank non hodgkins lymphoma. 7-9 An association between HCV infection and the syndrome of purpura, arthralgias, renal disease, and neuropathy was first reported in 1990 and accounts for approximately 60% to 80% of cases of mixed cryoglobulinemia.2 Conversely, the incidence of cryoglobulinemia in patients with chronic HCV infection has been variously reported as between 13% and 54%.10

Treatment of Mixed cryoglobulinemia associated with HCV: First-line therapy for mixed cryoglobulinemia due to HCV infection is antiviral therapy with pegylated interferon-? and ribavirin. Viral eradication usually produces marked reduction of complications and arrests end organ damage along with the clearance of cryoglobulins. Immunomodulators such as corticosteroids and cyclophosphamide are efficacious for symptomatic treatment of HCV cryoglobulinemia but may enhance viral replication. Prolonged therapy with these agents should be limited to severe vasculitis or aggressive glomerulonephritis who have failed to respond to antiviral therapy. In acute, fulminant presentations, plasmapheresis may prevent the rapid progression of the disease so that additional therapy can be initiated. 11


  1. Gorevic PD. Cryopathies: cryoglobulins and cryofibrinogenemia. In: Frank MM, Austen KF, Claman HN, Unanue ER, eds. Samters Immunologic Diseases. 2 vol. 5 ed; 1995:951-974.
  2. Kallemuchikkal U, Gorevic PD. Evaluation of cryoglobulins. Arch Pathol Lab Med. 1999;123:119-25.
  3. Romaszko JP, Tridon A, Jouanel P, Subtil E, Dibet P, Betail G. [Detection and analysis of cryoglobulins: comparative study of a population of patients and one of healthy controls]. Pathol Biol (Paris). 1993;41:525-9.
  4. Campioli D, Ghini M, Mascia MT et al. Characterization of cryoglobulins: some remarks on methodology. Clin Exp Rheumatol. 1995;13 Suppl 13:S75-8.
  5. Gorevic PD, Galanakis D, Finn AFJ. Cryoglobulins. In: Rose NR, de Nacario EC, Folds JD, Lane HC, Nakamura RM, eds. Manual of Clinical Laboratory Immunology. 5 ed: ASM; 1997:217-223.
  6. Grey HM, Kohler PF. Cryoimmunoglobulins. Semin Hematol. 1973;10:87-112.
  7. Lamelin JP, Zoulim F, Trepo C. Lymphotropism of hepatitis B and C viruses: an update and a newcomer. Int J Clin Lab Res. 1995;25:1-6.
  8. Sansonno D, Cornacchiulo V, Iacobelli AR, Gatti P, Distasi M, Dammacco F. Hepatitis C virus infection and clonal B-cell expansion. Clin Exp Rheumatol. 1996;14 Suppl 14:S45-50.
  9. Dammacco F, Gatti P, Sansonno D. Hepatitis C virus infection, mixed cryoglobulinemia, and non-Hodgkin's lymphoma: an emerging picture. Leuk Lymphoma. 1998;31:463-76.
  10. Wong VS, Egner W, Elsey T, Brown D, Alexander GJ. Incidence, character and clinical relevance of mixed cryoglobulinaemia in patients with chronic hepatitis C virus infection. Clin Exp Immunol. 1996;104:25-31.
  11. Kayali Z, Labrecque DR, Schmidt WN. Treatment of hepatitis C cryoglobulinemia: mission and challenges. Curr Treat Options Gastroenterol. 2006;9:497-507.

Contributed by Nidhi Aggarwal, MD and Bruce Rabin, MD, PhD

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