Clinical use:
Cryoproteins are plasma proteins which precipitate on cooling below normal physiological temperatures. More specifically, cryoglobulins are immunoglobulins that exhibit the phenomenon of insolubility when cooled below 37° C. The classification proposed by Brouet included three main categories.

Type 1: Monoclonal Cryoglobulins
Immunocytomata may produce immunoglobulins which polymerize (precipitate or gel) in the cold, usually within an hour. The patient may, thereby, present long before the tumour itself can be found and 90% of patients will eventually declare a malignant disease. The monoclonal immunoglobulin is usually an IgM, although other isotypes may also be seen. There is always a tendency to the hyperviscosity syndrome.

Type 2: Mixed Cryoglobulins
These include a monoclonal immunoglobulin, usually IgM kappa, with rheumatoid factor activity directed against the Fc portion of the polyclonal IgG. Whilst some are associated with lymphoproliferative disorders and other haematological malignancies, the majority are idiopathic and form the group of so-called ‘essential mixed cryoglobulinaemias’.

Type 3: Mixed Cryoglobulins
The cryoprecipitate, which may take up to 48 hours to appear in vitro, consists of immune complexes (most often polyclonal antibodies, IgM, IgA, and even IgE against polyclonal IgG, but also against DNA, etc). The complexes lodge in vessel walls and fix fibrinogen and complement, causing vasculitis, synovitis, serositis, or glomerulonephritis. They are associated mainly with ‘aberrant immunity’, eg systemic lupus erythematosus, rheumatoid arthritis, Sjogren’s syndrome, sarcoid, leprosy, syphilis, cytomegalovirus and EB virus infections, post-streptococcal glomerulonephritis, etc. Up to 30% of type 3 cryoglobulins are idiopathic and included in the ‘essential mixed cryoglobulinaemia’ group, almost all of which are now known to be associated with Hepatitis C infection.

Approximate frequency of features eventually found in patients with symptomatic mixed cryoglobulins

Purpura 90
Arthralgia 60
Infections 35
Ulceration/ necrosis 30
*Raynaud’s phenomenon 30
Abdominal pain 10
Paraesthesiae 10
Proteinuria/ renal failure 40
Hepatic involvement 35
Splenomegaly 30
Lymphadenopathy 15
Sjogren’s syndrome 15

*Of all patients presenting with Raynaud’s phenomenon only 7% have detectable cryoproteins.

Cryofibrinogen arises either from misdirected synthesis or from post-synthetic alteration. It is precipitated by heparin (which can be used for treatment) and is detected by comparing plasma, anticoagulated with oxalate or EDTA, and serum at 4° C. The precipitate will occur only in the plasma. Cryofibrinogenaemia can produce either skin lesions or thrombophlebitis migrans (when the underlying lesion is usually cancer).

Cryoprotein studies are indicated in any patient showing clinical manifestations which include intolerance of cold with pain in exposed areas, Raynaud’s phenomenon, and skin manifestations including purpura, urticaria, and ulcers. In 10% of patients with cryoglobulinaemia symmetrical neurological lesions occur. The most serious complication is renal failure which may occur in 40% of patients and requires therapeutic intervention.

Where, clinically, skin lesions produced by exposure to cold are flat and, when severe, gangrenous, cryoglobulins of Types 2 or 3, or cryofibrinogens are likely to be present. Where the skin lesions better conform to a nodular vasulitis, investigations for the presence of immune complexes may be considered. Such mixed cryoglobulins are the commonest aggregates which can be shown to precipitate in the cold.

Sample requirement:
5 mL serum. The samples must be taken into a warm syringe, needle and the blood collection tubes and separated at 37C.
5 mL EDTA plasma collected as for serum.
25 mL of urine (aliquot of 24 hour or early morning sample).

In general, cryoprecipitation is reversible with resolution at 37C but there are occasional proteins which do this badly. The blood must be kept at 37C until separation to avoid the cryoprecipitate being lost in the clot. The submission of both serum and EDTA plasma is essential for the full investigation of cryoproteins as some cryoproteins are known to require the presence of Ca ions to achieve polymerisation and precipitation.

Since heparin, itself, precipitates cryofibrinogen, this form of anticoagulation is contraindicated.

Centres offering this assay:
London St George’s Hospital PRU Diagnostic Service
Sheffield Northern General’s PRU Diagnostic Service

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