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Cryoglobulins

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Cryoglobulin Types

Cryoglobulins are abnormal immunoglobulins which form complexes and precipitate out of serum at low temperatures and resolubilise on warming. Other cryoprecipitable plasma proteins include cryofibrinogen and C-reactive protein-albumin complexes, but the vast majority are immunoglobulins. Minor alterations in the amino acid sequence of these immunoglobulins result in altered electrostatic interactions and solubility at low temperatures. The temperature at which cryoglobulins precipitate is influenced by their concentration: at higher concentrations, the temperature required for precipitation is also raised. IgM cryoglobulins tend to precipitate at lower temperature than do IgG cryoglobulins.

Purification and immunochemical characterisation of cryoglobulins has led to their classification into three major groups, each with recognised clinical associations.

Type I:	Single Monoclonal Immunoglobulin Type I cryoglobulins consist of a single monoclonal immunoglobulin, usually IgG or IgM, infrequently IgA, and very rarely monoclonal light-chain protein.
Type II:	Mixed Monoclonal/Polyclonal Immunoglobulins Type II cryoglobulins are mixed cryoglobulins consisting of two or more immunoglobulins of different classes. One component of the complex is a monoclonal protein (with a high proportion being IgM), that has rheumatology factor activity, in association with polyclonal IgG component.
Type III:	Mixed Polyclonal Immunoglobulins Type III cryoglobulins are also mixed cryoglobulins consisting of two or more immunoglobulins of different classes, however, each component is a polyclonal immunoglobulin.
Table 1: Cryoglobulin types and associated diseases
Type of Cryoglobulin		Immunochemical Composition	Associated Diseases	Comments
Type 1 : Single monoclonal immunoglobulins		IgM IgG IgA Monoclonal light-chains	B-cell dyscrasias: Myeloma Waldenstrom’s Macroglobulinaemia Chronic Lymphocytic Leukaemia Angioimmunoblastic Lymphadenopathy Hairy Cell Leukaemia	Cryoglobulin concentration is usually high.
Type II : Mixed monoclonal immunoglobulins		IgM-IgG IgG-IgG IgA-IgG	B-cell dyscrasias: Myeloma Waldenstrom’s Macroglobulinaemia Chronic Lymphatic Leukaemia Mixed essential Cryoglobulinaemia	Circulating immune complexes Cryoglobulin concentrations usually (>5g/L) Mainly associated with monoclonal paraproteinaemia
Type III : Mixed polyclonal immunoglobulins		IgM-IgG IgM-IgG-IgA	Autoimmune Disease: Lupus (SLE) Rheumatoid arthritis Scleroderma Sjogren’s Syndrome Chronic active hepatitis >Primary Biliary Cirrhosis. Autoimmune Chronic Active Hepatitis. Post streptococcal nephritis. Vasculitis Infections: EBV, CMV, Hepatitis, Leprosy, Kala-azar, tropical splenomegaly syndrome, >Lyme disease, Syphilis, Infective endocarditis	Cryoglobulin concentration is usually <1ml/mL. (<1%) Indicative of circulating immune complexes in response to antigen challenge in rheumatoid diseases and chronic infections.

Although there is considerable overlap, the majority of Type III cryoglobulins are present at low concentration, with a cryocrit <1%. Type I and Type II cryoglobulins are often present at a higher concentration, with a cryocrit usually >1%, and occasionally up to 20-40%.

Clinical Presentation

The commonest symptoms of cryoglobulinaemia are skin lesions, which are found in up to 80% of patients. Arthralgia and/or arthritis (35%). Glomerulonephritis producing nephrotic syndrome and/or hypertension (20%) and neurologic symptoms (17%) including stroke, peripheral neuropathy and mononeuritis multiplex are also common symptoms (Table 2).

All types of cryoglobulins may be responsible for specific symptoms that occur in response to cold weather, such as Raynaud’s phenomenon, vascular purpura, bleeding tendencies, cold-induced urticaria, and even distal arterial thrombosis with gangrene. Since Type II and Type III cryoglobulins are circulating soluble immune complexes, they may be associated with a serum-sickness-like syndrome characterised by polyarthritis, vasculitis, glomerulonephritis, or neurological symptoms. In patients with mixed essential cryoglobulinaemia (MEC), a syndrome may occur that is associated with arthralgias, palpable purpura, weakness, and frequently lymphadenopathy and hepatosplenomegaly. Glomerulonephritis (sometimes rapidly progressive) is common in MEC. The MEC syndrome is often a sequela of infection with Hepatitis B or Hepatitis C virus, and may respond to interferon therapy.

Type I cryoglobulins are more likely to be symptomatic, and are usually associated with acrocyanosis, retinal haemorrhage, Raynaud’s phenomenon, and arterial thrombosis. High level of Type I cryoglobulins are associated with symptoms of hyperviscosity. Mixed cryoglobulins, on the other hand, are less commonly symptomatic, and are usually associated with vascular pupura and athralgias/arthritis. This is secondary to deposition within tissues of what are essentially immune complexes which activate complement and induce localised inflammation.

Detection of Cryoglobulins

The thermolability of cryoglobulins necessitated that the blood samples be collected at 37^oC. Syringes and collection tubes must be at 37^oC at the time of blood collection and maintained at 37^oC until clotting is completed. Tubes for collection should not be anticoagulated, since the use of plasma may result in the development of cold-precipitable fibrinogen (cryofibrinogen) or heparin-precipitable protein.

Collection Procedures

1. Collect 10mL of blood in a warm syringe (37^oC).
2. Fill a specifically designed glass cryoglobulin tube that has been warmed to 37^oC with a minimum volume of 6mL of blood.
3. The tube must remain at 37^oC until the blood clots.
4. The serum is separated from the clot by centrifugation at 37^oC and then stored at 4^oC. Do not centrifuge at temperatures below 37^oC.

Analysis Process

1. A white precipitate (cryoglobulins) appears in the serum after 24-72 hours of storage at 4^oC.
2. The serum is tested for reversibility of the cryoprecipitate by rewarming an aliquot at 37^oC for 24 hours.
3. The cryoglobulin can be quantitated by:

• measuring the serum protein concentration before and after cryoprecipitation
• measuring the serum immunoglobulin concentrations before and after cryoprecipitation.
• isolating the precipitate from an aliquot of serum, dissolving it in an acidic buffer and measuring the cryoglobulin protein level spectrophotometrically by absorbance at 280mm.

4. After isolating and washing the cryoprecipitate, the identity of the components of the cryoglobulin can be determined by immunoelectrophoresis or by isoelectric focusing in association with immunofixation. These tests must be performed at 37^oC to avoid precipitation and hence loss of the cryoglobulin during the procedures.

Interference with Laboratory Tests

The presence of cryoglobulins which precipitate at ambient temperatures and thereby remove serum components may affect various laboratory tests. The most noteworthy example is complement fixation and immunoglobulin entrapment and inactivation, resulting in falsely low readings for complement activity and immunoglobulin levels. These are often not fully restored by redissolving the cryoprecipitate with warming.

False Negative Results

False negative results for cryoglobulins will occur if:

• anticoagulated tubes are used for specimen collection.
• the syringe is not warmed to 37^oC.
• the sample is not kept at 37^oC until clotting is completed.
• the sample is centrifuged at temperatures below 37^oC.
• the sample is not stored at 4^oC for 72 hours.

References

1. BROUET JC et al. Biologic and clinical significance of cryoglobulins: a report of 86 cases. Am J Med 1974, 57:755-788.
2. FEINER HD. Relationship of tissue deposits of cryoglobulin to clinical features of mixed cryoglobulinaemia. Hum Pathol 1983, 14:71-715.
3. GOREVIC PD et al. Mixed cryoglobulinaemia: clinical aspects and long-term follow-up of forty patients. AmJMed 1980, 69:287-308.
4. LEVO Y et al. Interferon a2a therapy in cryoglobulinaemia associated with hepatitis C virus. N Engl J Med 1994, 330:751-754.
5. MALCHESKY PS, CLOUGH JD. Cryoimmunoglobulins: Properties, prevalence in disease, and removal. Cleve Clin 1985; 52: 175-192.
6. DENMAN AM. Cryoglobulins and the immunopathological manifestations of autoimmune disease. Clin Exp Immunol 1992; 87: 169-171.
7. WINFIELD JB. Cryoglobulinaemia. Hum Pathol 1983; 14: 350-354.
8. LEVEY JM et al. Mixed cryoglobulinaemia in chronic Hepatitic C infection. Medicine. 1994; 73: 53-67.
9. GALA S. Cryoglobulins. ASCIA Newsletter. Issue 12. March/April 1996; page 13-15.

Written by:                     Glenn Reeves and Maree Gleeson, Immunology, HAPS

Last Reviewed:             30 November 1998