1. Practical Blood Bank
Lab 11
Cyroglobulin

2. *
07/16/96
Cryoglobulins
Cryoglobulins are immunoglobulins that precipitate reversibly at lowered temps.
The temperature ranges from degrees.
Cryoglobulin concentrations are expressed as percent . *

3. 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.

4. 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.

5. 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.

6. 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.

7. Type I Cryoglobulin Disorders
Examples of Type I Cryoglobulins disorders include
Waldenstrom’s Macroglobulinemia,
Paroxysmal Cold Hemoglobinuria,
and Idiopathic Nonmalignant Monoclonal Cryoglobulinemia.

8. 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.
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.

9. 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.
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 degrees C. When the temperature rose, red blood cells lysed. The antibody specificity is directed against the red blood cell antigen P3.

10. 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.
Age groups most susceptible for PCH are the elderly population between the ages of years old.

11. 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.

12. 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

13. Note: Essential Mixed Cryoglobulinemia or EMC is slightly more common in females than males. In fact, females that are between the ages of years of age are more susceptible.

14. Treatment of Type II Cryoglobulin Disorders
Treatment for EMC have involved the following:
Plasma Exchange
Steroids
Use of Cytotoxic Drugs

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

16. *
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. *

17. 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.

18. 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

19. Put one tube in the refrigerator for a minimum of 4 days
Leave the other tube at room temperature

20. Quality Control A known positive patient sera may be used as
a positive control when available. There is no
commercial material available.

21. Test Results
Report results as either positive or negative. A negative Cryoglobulin is considered normal.
Negative
(at 4C)
Positive
(at 4C)

22. 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.

23. 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

24. 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.

25. 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.

26. 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.

27. 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.