CryoglobulinOutline I. Cryoglobulins Background II. Types of antibody classes associated with Cryoglobulins III. The diseases associated with Cryoglobulins IV. Cryoglobulin disease treatments V. The Cryoglobulin test principle
Cryoglobulins Outline (Cont.) VI. The Preparation of Specimen VII. The Collection Procedure VIII. The Cryoglobulin Test Procedure IX. Quality Control Test X. Test Results XI. Conclusion
CryoglobulinsBackground • Cryoglobulins are immunoglobulins that precipitate reversibly at lowered temps. • The temperature ranges from 0 - 4 degrees. • Cryoglobulin concentrations are expressed as percent of total volume.
Cryoglobulins Background (Cont.) Recent studies have shown that the temperature at which cryoglobulins precipitate varies with the total protein concentration. Higher concentrations of protein in a sample increases the temperature at which the cryoglobulins precipitate.
Cryoglobulins Background (Cont.) Cryoglobulins are made up of monoclonal antibodies IgM or IgG, rarely IgA. IgM tends to precipitate at lower temperatures than does IgG cryoglobulin.
Cryoglobulins Background (Cont.) Occasionally, IgM macroglobulin is both cryo precipitable and capable of cold induced antibody mediated agglutination of red cells.These are referred to as cold agglutinins. Not all cryoglobulins are cold agglutinins because they do not share some of the antibody characteristics of cold agglutinins.
Types of Cryoglobulins Cryoglobulins are classified into three types based on their composition. Type I - is made up of a monoclonal single homogeneous immunoglobulin usually IgM or IgG. The Cryoglobulin concentration is usually high, greater than 5 mg/ml.
Types of Cryoglobulins (Cont.) Type II - are classified as mixed cryoglobulins • composed of a monoclonal component usually IgM and a polyclonal component IgG. IgM is associated with a rheumatoid factor activity. Concentrations are usually greater than 1 mg/ml.
Types of Cryoglobulins (Cont.) Type III - are mixed cryoglobulins that lack a monoclonal component. • Type III consists of two or more immunoglobulins of different classes that are polyclonal immunoglobulins. Their concentration is usually less than 1mg/ml.
Type I Cryoglobulin Disorders Examples of Type I Cryoglobulins disorders include Waldenstrom’s Macroglobulinemia, Paroxysmal Cold Hemoglobinuria, and Idiopathic Nonmalignant Monoclonal Cryoglobulinemia.
Type I Cryoglobulin Disorders (Cont.) Symptoms of Type I Waldenstrom’s Macroglobulinemia include hyperviscosity of the blood, cold urticaria, Raynaud’s Phenomenon, Purpura, and Cutaneous Vasculitis with or without ulcerative retinal hemorrhage. These monoclonal antibodies are IgM or IgG.
Type I Cryoglobulin Disorders (Cont.) In the case of Waldenström's Macroglobulinemia, these types of cryoglobulins precipitate due to cold exposure and may induce partial or complete occlusion of small blood vessels.
Type I Cryoglobulin Disorders (Cont.) Idiopathic nonmalignant monoclonal cryoglobulinemia may be related to a unique gene arrangement in certain individuals. Studies have shown that this may relate to the high rate of synthesis of cryoglobulins in nonmalignant B cells.
Type I Cryoglobulin Disorders (Cont.) Paroxysmal Cold Hemoglobinuria is caused by a Type I cryoglobulin that lyses red blood cells at 37 degrees Celsius. The key point is that compliment was attached at 0 - 4 degrees C. When the temperature rose, red blood cells lysed. The antibody specificity is directed against the red blood cell antigen P3.
Type I Cryoglobulin Disorders (Cont.) Symptoms of (PCH) include, discoloration of the area varying from pale gray to dark blue- violet. Usually the areas are the fingers, toes, earlobes, and the nose. Pain and numbness is associated with PCH as well as anemia.
Type I Cryoglobulin Symptoms Age groups most susceptible for PCH are the elderly population between the ages of 60 - 80 years old.
Treatments for Type I Cryoglobulin Disorders Treatment for Type I Cryoglobulin disorders are designed to eliminate the immune complexes between Cryoglobulin antibodies and antigens. This can be done by prewarming of blood in transfusions, and administering of steroids. Splenectomy has been performed in selected idiopathic cases.
Type II Cryoglobulin Disorders These disorders are associated with a major disorder called Essential Mixed Cryolobulinemia or (EMC).
Type II Cryoglobulin Disorders (Cont.) The symptoms for the Type II Cryoglobulin disorder, EMC include the following: • Purpura • Weakness • Arrhythmia • Hepatosplenomegaly • Glomerulonephritis
Type II Cryoglobulin Disorders (Cont.) Note: Essential Mixed Cryoglobulinemia or EMC is slightly more common in females than males. In fact, females that are between the ages of 30 - 50 years of age are more susceptible.
Treatment of Type II Cryoglobulin Disorders Treatment for EMC have involved the following: • Plasma Exchange • Steroids • Use of Cytotoxic Drugs
Type III Cryoglobulins Disorder Type III Cryoglobulins are very rare occurrences and occur in low concentrations. They are usually resolved when the precipitate dissolves.
Cryoglobulin Test Principle Cryoglobulins are abnormal immunoglobulins that form complexes and precipitate out of serum at low temperatures and redissolve upon warming or returning to room temperature. The Cryoglobulin test detects antibodies in the blood that may cause sensitivity to low temperatures.
Specimen Preparation The specimen required is whole blood. The sample is brought to the laboratory immediately after drawing. The specimen must not be refrigerated before the test.
Specimen Preparation (Cont.) . Tubes for collection should not be anticoagulated blood since the use of plasma may result in the development of cold-precipitable fibrinogen, (cryofibrinogen) or heparin-precipitable protein.
Collection Procedures • Collect 10 ml of blood in a red top tube • The specimen must be incubated for at least 30 minutes to 1 hour at 37 degrees C in a heat block or water bath prior to centrifugation • Centrifuge at room temperature • Transfer the serum to two test tubes labeled “room temperature” and “refrigerator”
Cryoglobulin Test Procedure • Place fresh serum into the appropriately labeled tube • Put one tube in the refrigerator for a minimum of 4 days • Leave the other tube at room temperature
Quality Control A known positive patient sera may be used as a positive control when available. There is no commercial material available.
Test Results • Report results as either positive or negative. A negative Cryoglobulin is considered normal.
Interpretation of Results • A positive test is indicated by a strong cloudy or turbid sample. If cloudiness is present, place tube in 37C water bath for 2 hours. If cloudiness remains, it is due to something other than cryoglobulin. • A negative test is indicated by a clear, non-turbid sample. This means Cryoglobulins are not present.
Results Positive Negative (at 4C) (at 4C)
False Negative Results • The syringe is not warmed to 37 degrees C. • The sample is not kept at 37 degrees C until clotting is completed • The sample is centrifuged at temperatures below 37 degrees C • The sample is not stored at 4 degrees C for 72 hours
False Positive Results • If any lipemia is present, this must be taken into consideration, lipemia is not a sign of cryoglobulinemia. This would be a false positive. • Anticoagulated tubes are used for specimen collection
Summary • Cryoglobulins are antibodies that precipitate or become insoluble at cold temperatures but become soluble when plasma is rewarmed. • There are 3 types of cryoglobulins. • The predominate monoclonial cryoglobulin class is mainly IgM. • IgM is considered a complete or cold antibody.
Summary, (Cont.) Positive test results of less than 1 mg/ml is considered normal. Positive test results of concentrations of Cryoglobulins greater than 1 mg/ml is indicative of disease. If positive, the exact composition of protein is detected by immunofixation electrophoresis.
Summary, (Cont.) The test for detecting cryoglobulins is based on the effects of temp and their solubility.