Cryoglobulins

1
Cryoglobulins
2


Cryoglobulin Outline

• 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

3
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

4
Cryoglobulins Background

• 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.

5
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.

6
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.

7
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.

8
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.

9
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.

10
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.

11
Type I Cryoglobulin Disorders

• Examples of Type I Cryoglobulins disorders
• include Waldenstroms Macroglobulinemia,
• Paroxysmal Cold Hemoglobinuria, and
• Idiopathic Nonmalignant Monoclonal
• Cryoglobulinemia.

12
Type I Cryoglobulin Disorders (Cont.)

• Symptoms of Type I Waldenstroms
• Macroglobulinemia include hyperviscosity of
• the blood, cold urticaria, Raynauds
• Phenomenon, Purpura, and Cutaneous
• Vasculitis with or without ulcerative retinal
• hemorrhage. These monoclonal antibodies
• are IgM or IgG.

13
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.

14
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.

15
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.

16
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.

17
Type I Cryoglobulin Symptoms

• Age groups most susceptible for PCH are the
  elderly population between the ages of 60 - 80
  years old.

18
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.

19
Type II Cryoglobulin Disorders

• These disorders are associated with a major
  disorder called Essential Mixed Cryolobulinemia
  or (EMC).

20
Type II Cryoglobulin Disorders (Cont.)

• The symptoms for the Type II Cryoglobulin
• disorder, EMC include the following
• Purpura
• Weakness
• Arrhythmia
• Hepatosplenomegaly
• Glomerulonephritis

21
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.

22
Treatment of Type II Cryoglobulin Disorders

• Treatment for EMC have involved the following
• Plasma Exchange
• Steroids
• Use of Cytotoxic Drugs

23
Type III Cryoglobulins Disorder

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

24
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.

25
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.

26
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.

27
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

28
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

29
(No Transcript)
30
(No Transcript)
31
Quality Control

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

32
Test Results

• Report results as either positive or negative. A
  negative Cryoglobulin is considered normal.

33
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.

34
Results

• Positive

Negative (at 4C)
(at 4C)
35
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

36
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

37
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.

38
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.

39
Summary, (Cont.)

• The test for detecting cryoglobulins is based
• on the effects of temp and their solubility.