Practical Blood Bank Lab 11 * * 07/16/96 * ## * 07/16/96 - PowerPoint PPT Presentation

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Practical Blood Bank Lab 11 * * 07/16/96 * ## * 07/16/96

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Practical Blood Bank Lab 11 * * 07/16/96 * ## * 07/16/96 * ## Cryoglobulins Cryoglobulins are immunoglobulins that precipitate reversibly at lowered temps. – PowerPoint PPT presentation

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Title: Practical Blood Bank Lab 11 * * 07/16/96 * ## * 07/16/96


1
Practical Blood Bank
Cyroglobulin
Lab 11
2
Cryoglobulins
  • Cryoglobulins are immunoglobulins that
    precipitate reversibly at lowered temps.
  • The temperature ranges from 0 - 4 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
  • Waldenstroms Macroglobulinemia,
  • Paroxysmal Cold Hemoglobinuria,
  • and Idiopathic Nonmalignant Monoclonal
    Cryoglobulinemia.

8
  • 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.
  • 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 0 - 4 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 60 - 80
    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 30 -
    50 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
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 14, 18etc.
  • 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 132 to 164 in a single convalescent
    specimen are significant.
  • Low titers of 116 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.
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