Cryoglobulins

Cryoglobulins Classification and diagnosis

Sample for cryoglobulins

1. Venous blood is needed.
2. Keep the blood at 37 °C.
3. Separate the serum at 37 °C, so the cryoglobulins remain in the serum.

Precautions for detecting the cryoglobulins

1. Maintain the temperature of the blood at 37 °C.
2. Also, maintain the temperature of the serum at 37 °C.

Purpose of the test (Indications) for detecting the cryoglobulins

1. This test is done to diagnose:
   1. Purpura.
   2. Arthralgia.
   3. Raynaud phenomenon.

Pathophysiology of Cryoglobulins

Definition:

1. These immunoglobulins precipitate in serum reversibly at low temperatures or may gel at cold temperatures.
2. Most cryoglobulins are polyclonal immunoglobulin complexes, and nearly half are monoclonal, usually IgM.
3. Cryoglobulins are abnormal immunoglobulins proteins produced due to various diseases.

Cryoglobulins properties:

1. These are insoluble at 4 °C.
2. May aggregate up to 30 °C.
3. It can fix complement.
4. It can initiate an inflammatory response.

Cryoglobulinemia:

1. It is defined as a condition where plasma or serum proteins (IgM) precipitate when cooled below body temperature.
2. These proteins precipitate at 4 °C and dissolve again at 37 °C.
3. These are the proteins that reversibly precipitate or gel at 4 °C.

Cryoglobulins main features

These cryoglobulins are classified as follows:

1. Type I (Monoclonal).
2. Type II (Mixed cryoglobulin, a mixture of polyclonal immunoglobulins).
3. Type III (Polyclonal).
   1. Primary or idiopathic or essential.
   2. Secondary that is associated with diseases.

Type I monoclonal cryoglobulins:

1. These are produced by the neoplastic lymphocytes and plasma cells. These are typically IgM types of immunoglobulin.
   1. Associated with multiple myeloma, macroglobulinemia, and rare neoplasm of the plasma cells and lymphocytes.
   2. Blood may gel when drawn.
   3. Sometimes these are idiopathic.
   4. May see IgG as well.
   5. Rarely see IgA only.
2. These may constitute 25% of the cryoglobulins.
   1. Often present in large amounts >5 mg/dL in the serum.
3. Symptoms are severe, and often there may be gangrene without any other cause.
   1. The patient may have Raynaud’s syndrome-like symptoms.

Type II cryoglobulins:

1. These are monoclonal immunoglobulins mixed with one type of polyclonal immunoglobulins; the most common is IgM and polyclonal IgG, always seen with RF.
2. It is seen in 25% of the cases.
3. These consist of two types:
   1. The monoclonal form is seen as the rheumatoid factor and usually is IgM with a light kappa chain.
   2. The second is polyclonal IgG which is always seen as a Rheumatoid factor (RF).
   3. RF may be seen as a high titer without the disease.
   4. It is most often seen with HCV infection and less often with HBV, EBV, and other diseases like bacterial infection, parasitic infestation, autoimmune disorders, Sjogren’s syndrome, and immune complex nephritis.
4. C4 level is decreased.

Type  III  cryoglobulins:

1. These are mixed cryoglobulins and the most common combination of IgG and IgM (usually with RF).
2. It is seen in ∼50% of the cases.
3. It usually occurs in a small amount of <1 mg/dL in the serum.
4. More than 90% contain IgM, rheumatoid factor, and IgG.
5. Most commonly seen with lymphoproliferative disorders and connective tissue diseases like SLE.
6. These are also seen in patients with persistent HCV infection.
   1. Mostly seen in autoimmune diseases.
7. There are circulating immune complexes produced by various antigens like viral, bacterial, and autologous antigens.

Mixed cryoglobulinemia:

1. It consists of type II and type III.

Signs and symptoms of Cryoglobulins:

1. Cryoglobulinemia is a systemic immune-complex disease characterized by clinical syndrome-like:
   1. Palpable purpura. This is a constant feature and is present in the lower extremities.
   2. Arthralgias.
   3. Hepatosplenomegaly.
   4. Lymphadenopathy.
   5. Anemia.
   6. Glomerulonephritis. The most common is membranoproliferative glomerulonephritis ( MPGN type II).
   7. Most patients are hypocomplementemic, reflecting immune-complex disease.
2. These proteins precipitate in the blood vessels of fingers when exposed to cold.
3. These patients will have symptoms of:
   1. Vascular purpura. This is the most common symptom and is seen in 60% to 100% of the patients.
   2. Bleeding tendency.
   3. Arthralgia. It is the second most common, most seen in 60% to 90% of the cases.
   4. Cold-induced urticaria.
   5. Raynaud phenomenon (pain, cyanosis, the coldness of fingers). It is seen in 50% of the cases.
4. These proteins are present in various diseases.
   1. Serum level > 5 mg/dL = Multiple myeloma, macroglobulinemia, and leukemia.
   2. Serum level between 1 to 5 mg/dL= Rheumatoid arthritis.
   3. Serum level < 1 mg/dL = SLE, RA, infectious mononucleosis, viral hepatitis, endocarditis, cirrhosis, glomerulonephritis.

Normal Cryoglobulins:

• The serum is negative for cryoglobulins.

Procedure for Cryoglobulins:

1. Take the blood and keep it at 37 °C until it is clotted.
2. Clotted blood is centrifuged at 37 °C.
3. Keep the serum in the fridge at 4 °C.
   1. Please keep it for at least 3 days and again centrifuge at 4 °C.
4. Check the serum daily for white precipitate or gel.
   1. Checking the time period is disputed; some suggest 3 days, while others propose 7 days.
5. Check daily for at least 72 hours to 7 days.
   1. The precipitate is recorded and measured, which is cryoglobulin.
   2. Rewarm the sample if it dissolves, then it confirms the cryoglobulins.
6. Cryoprecipitate is washed and subject to Immunoelectrophoresis to identify the type of cryoglobulin (immunoglobulin).

Another procedure for cryoglobulin is:

1. Collect the blood in EDTA or citrate and also make a serum sample.
2. Incubate both at 4 °C.
3. A precipitate in the plasma but not in the serum will indicate cryoglobulins.

Causes of Cryoglobulins:

1. Autoimmune diseases like SLE, RA, and Sjogren’s syndrome.
2. Malignancies like Multiple myeloma, Leukemia, Waldenstrom macroglobulinemia, and Lymphoma.
3. Acute and chronic infections like Infectious mononucleosis, Poststreptococcal glomerulonephritis, and endocarditis.
4. Liver diseases like Cirrhosis and Hepatitis.
5. Renal involvement manifests as either nephrotic or nephritic syndrome associated with hypocomplementemia.
   1. The most common finding is membranoproliferative glomerulonephritis type II.

Treatment of cryoglobulins:

1. It is attempted on immune modulation using corticosteroids, especially in mixed types of cryoglobulins.
2. Can try plasmapheresis.
3. α-interferon in patients with HCV.
4. Remissions are achieved in 75% of the cases of mixed type.
   1. Relapse is seen in 50% of the cases in mixed type.

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