Cryoglobulins

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
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
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.
 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.