Cryoglobulins

Cryoglobulins are immunoglobulins that precipitate in serum at temperatures under 37 °C and redissolve on rewarming.

They are classified by Brouet: type I (monoclonal immunoglobulin alone, usually IgM or IgG, associated with lymphoproliferative disease), type II (monoclonal IgM with rheumatoid factor activity bound to polyclonal IgG, mixed cryoglobulinaemia, usually hepatitis C related), and type III (polyclonal IgM and IgG, associated with infection and autoimmune disease).

Types II and III are called mixed cryoglobulinaemia. The test involves collecting and clotting blood at 37 °C, separating the serum warm, storing it at 4 °C for up to 7 days, and measuring the cryocrit (percentage packed precipitate volume) and characterising the precipitate by immunofixation.

Cryoglobulinaemic vasculitis is a small vessel immune complex vasculitis with purpura, arthralgia, weakness (the Meltzer triad), peripheral neuropathy, membranoproliferative glomerulonephritis and skin ulcers. Hepatitis C causes 70 to 90% of mixed cryoglobulinaemia, and about 30 to 50% of people with chronic hepatitis C have circulating cryoglobulins, though only 5 to 10% develop vasculitis.

Other causes are hepatitis B, HIV, Sjögren syndrome, SLE, rheumatoid arthritis, lymphoma, myeloma, Waldenström macroglobulinaemia and chronic infections. Essential mixed cryoglobulinaemia (no cause) is now uncommon.

Indications. Palpable purpura, especially of the lower limbs. Unexplained arthralgia with weakness. Glomerulonephritis with low C4 and positive rheumatoid factor. Peripheral neuropathy with skin lesions. Chronic hepatitis C with extrahepatic features. Hyperviscosity, Raynaud phenomenon and digital ischaemia on cold exposure (type I).

Known lymphoproliferative disease with cold related symptoms. Spuriously low platelet count or leucocyte count on automated analyser (cryoglobulin precipitation).

Specimen. Venous blood, 10 to 20 mL, collected into a pre-warmed (37 °C) dry glass or plain tube (no gel and no anticoagulant) with a syringe and needle also warmed, and transported at 37 °C (in a flask of warm water or a heat block). Clot at 37 °C for 1 to 2 hours, centrifuge warm, and transfer serum to a clean tube for storage at 4 °C for 7 days. Errors in this process are the commonest cause of false negatives.

Reference values.

• Negative: no cryoprecipitate after 7 days at 4 °C.
• Cryocrit: normally absent, and reported as percentage (under 1% is trace).
• Type I: often high cryocrit (over 5%), monoclonal immunoglobulin alone.
• Type II: cryocrit 1 to 70%, monoclonal IgM-kappa with polyclonal IgG, rheumatoid factor positive.
• Type III: low cryocrit (under 5%), polyclonal IgM and IgG.

Interpretation.

• Type I: associated with Waldenström macroglobulinaemia, multiple myeloma and monoclonal gammopathy. Presents with hyperviscosity and occlusive (not inflammatory) vascular lesions: livedo, acral necrosis, ulcers, Raynaud phenomenon, retinal haemorrhage and neurological symptoms.
• Type II (mixed): hepatitis C, Sjögren syndrome, lymphoproliferation. Presents with immune complex vasculitis, with low C4, positive rheumatoid factor, and renal and neurological involvement.
• Type III (mixed polyclonal): infection, SLE, rheumatoid arthritis, Sjögren syndrome, hepatitis, and often asymptomatic.
• Low C4 (often profound) with a normal or mildly low C3, and strongly positive rheumatoid factor, are characteristic of type II cryoglobulinaemia.
• Cryoglobulins without symptoms are common in hepatitis C, and require monitoring rather than treatment.
• Cryocrit does not always correlate with severity.

Critical values and findings needing immediate action.

• Purpura with rapidly progressive glomerulonephritis and low C4: severe cryoglobulinaemic vasculitis, urgent nephrology review, immunosuppression (rituximab, high dose steroids), plasma exchange, and antiviral therapy for hepatitis C.
• Digital ischaemia, skin necrosis or stroke with a high cryocrit (type I): hyperviscosity syndrome, plasma exchange and treatment of the underlying clone.
• Mononeuritis multiplex: urgent immunosuppression.
• Suspected cryoglobulinaemia in hospital: the sample is kept at 37 °C, and cold exposure (cold fluids, unheated transfusion) is avoided.

Interferences and pitfalls.

• Sample handling is critical. If blood cools before clotting and centrifugation, cryoglobulins are lost in the clot and the result is falsely negative. If in doubt, the test is repeated with correct technique (three consecutive samples are often advised in highly suspected cases).
• Anticoagulated samples (EDTA, heparin) can cause false positive precipitates (cryofibrinogen, which is detected in plasma but not serum).
• Reading time: cryoglobulins in type III may take up to 7 days to precipitate, and early reading underestimates.
• Cryocrit and clinical severity are not tightly linked, and complement (C4), rheumatoid factor and clinical features direct management.
• Cold agglutinins cause agglutination of red cells, which is a different phenomenon.
• Cryoglobulin typing requires immunofixation, which shows monoclonal heavy and light chains.
• Hepatitis C RNA testing, rather than antibody alone, shows active infection and guides antiviral therapy.
• Spurious laboratory results (pseudothrombocytopenia, pseudoleucocytosis) are a clue when the analyser samples are not warmed.
• Cryofibrinogenaemia is a separate test, performed on plasma.

Special groups.

• Children: rare, and associated with infection.
• Pregnancy: rare, with flares of vasculitis, and direct acting antivirals are not used in pregnancy.
• Older adults: monoclonal gammopathy and lymphoproliferative disease are more frequent causes.
• Regions: hepatitis B and C, HIV, and chronic infections are common causes, and the direct acting antiviral treatment of hepatitis C can induce remission in many patients.

Follow up tests. Hepatitis C antibody and RNA, hepatitis B surface antigen and core antibody, HIV, rheumatoid factor, complement C3, C4 and CH50, serum protein electrophoresis and immunofixation, serum free light chains, immunoglobulins, full blood count, creatinine and urinalysis, liver function tests and fibrosis assessment, ANA and ENA, nerve conduction studies,

skin or renal biopsy, bone marrow biopsy if lymphoproliferation is suspected, and cross-sectional imaging for lymphoma.