Practical Blood Bank Lab 11 Cyroglobulin

* 07/16/96 Cryoglobulins Cryoglobulins are immunoglobulins that precipitate reversibly at lowered temps. The temperature ranges from 0 - 4 degrees. Cryoglobulin concentrations are expressed as percent . *

Cryoglobulins 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 are made up of monoclonal antibodies IgM or IgG, rarely IgA. IgM tends to precipitate at lower temperatures than does IgG cryoglobulin.

Cryoglobulins 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. 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 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 II Cryoglobulin Disorders These disorders are associated with a major disorder called Essential Mixed Cryolobulinemia or (EMC). The symptoms for the Type II Cryoglobulins disorder, EMC include the following: Purpura Weakness Arrhythmia Hepatosplenomegaly Glomerulonephritis

Type III Cryoglobulins Disorder Type III Cryoglobulins are very rare occurrences and occur in low concentrations. They are usually resolved when the precipitate dissolves.

* 07/16/96 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. 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” 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. Negative (at 4C) Positive (at 4C)

Interpretation of Results A positive test is indicated by a strong cloudy or turbid sample. If cloudiness is present, place tube in 37 C 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.

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 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

Cold Haemagglutination test Blood is allowed to clot at 37o C for 30 minutes and serum is separated immediately.  Refrigerating the sample of whole blood before separating serum may lead to false negative results.   Reagents Needed: 0.9 % Normal Saline. Commercial Blood RBC's contain most of RBC's antigens OR 2% suspension of human group O, the cells should not be more than 3 days old, and the suspension prepared freshly.

Procedures Place 10 tubes in a rack and mark from 1 to 10. Put 0.75 ml of 0.9% Saline in tube no.(1), and 0.5 ml of Saline into each of the remaining tubes. Add 0.25 ml of patient serum to tube no.(1), mix and transfer 0.5 ml to tube no.(2) , continue transferring the same way till tube no.(9), then discard 0.5 ml of the mixture. Tube no.(10) is antigen control (Cells). Dilutions 1:4, 1:8…etc. Add 0.05 ml of DiaCells I, II, III or Prepared Group O Cells to each tube. Mix the suspension and place in the refrigerator overnight at 0-5oC.

Remove the tubes from the fridge and Read for agglutination immediately macroscopically, because in Warming the antibody present may elute from the cells leading to false negative results. Titer: The highest serum dilution showing agglutination of RBC's Macroscopically.

Interpretation of results Titers of 1:32 to 1:64 in a single convalescent specimen are significant. Low titers of 1:16 are found in normal individuals. In Atypical Pneumonia 50-80% of patients may demonstrate a four-fold increase in titer between first and fourth week.