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Autoantibody Information and Courses from MediaLab, Inc.

These are the MediaLab courses that cover Autoantibody and links to relevant pages within the course.

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Antibody Detection and Identification (retired 12/6/2013)
Example Of An Autoantibody (warm)

The auto control and all panel cells are showing reactivity at the AHG phase, which indicates a possible autoantibody (warm).IS = Immediate Spin; AHG = Antihuman Globulin Phase; CC = Check Cells; AC = Auto Control; ND= Not done

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Cold antibodies

Most are IgM and not clinically significant May interfere with detection of clinically significant antibodies if they react at AHG phase. Screen cells and panel cells will have positive reactions in IS phase and strength will diminish or antibody will not be detected at AHG phase. Auto control will be positive if the cold antibody is an autoantibody. Binding of antibody to antigen occurs at room or colder temperatures and may start to disassociate from the red cell membrane at warmer temperatures. Reactions will appear weaker or be negative at warmer temperatures. (Example: 4+ at IS phase and W (weak)+ at AHG phase.) PrewarmingIf a non specific cold antibody or cold agglutinin is suspected, warm the sample and testing reagents, including saline, to 37° C. Only do reaction readings at AHG; bypassing the optimum reaction temperature prevents activation and binding of the cold antibody .

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Is It a Cold or a Warm Autoantibody?

Cold antibody Immediate spin screen and panel cell reactions will be positive (W+ to 4+). The auto control may also be positive. AHG reactions may be weakly positive if the cold antibody is bound strongly to the red cells. Prewarming should prevent binding from occurring. So, prewarm panels and tests should have negative reactions.Warm antibody Immediate spin screen, panel cell and auto control usually not positive. AHG reactions will be positive including auto control (W+ to 4+). Prewarming of sample and reagents will not change positive reactions since they react best at 37°C and AHG phase. So, reactions will still be positive. Elution and autoadsorption techniques may be used to help further identify the antibody or to help identify other clinically significant antibodies that may be present.AutoadsorptionAutoadsorption is a technique that involves adsorbing unbound autoantibody from the patient's serum using the patient's own red cells. Once the autoantibody is removed, then testing can be performed to determine if any clinically significant antibodies are present.

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What is an advanced technique that can help to determine the identity of other clinically significant antibodies that are present if a patient has a warm autoantibody?View Page
Cold Autoantibody Example

Reactions are occurring strongly in IS phase but are weaker in AHG phase which could be due to some disassociation of the cold antibody occurring at the warmer testing phase.Prewarming of all reagents and sample will prevent binding of cold antibody. If the W+ reactions at AHG are due to residual cold antibody, the reactions should be negative with a prewarmed panel. No IS phase reading is performed. Prewarmed Panel

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When to Suspect an Autoantibody

Autoantibody (Cold or Warm)Suspect an autoantibody if: All panel and screen cells are positive and auto control is positive. Reaction strength in all the cells will be the same.Cold autoantibody: strongest reactions occur at immediate spin phase. Reactions may weaken in strength at 37°C and AHG.Warm autoantibody: strongest reaction occurs at AHG. Reactions may be seen at 37°C and usually not seen at immediate spin.

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Immune Antibodies

Immune antibodies occur in the serum of individuals who become sensitized to foreign antigens through pregnancy or transfusion. IgM predominates in the primary response, IgG in the secondary response. Most react at 37°C and are considered clinically significant. Examples include antibodies in the Kell, Rh, Duffy, and Kidd systems. Immune antibodies can be classified as alloantibodies or autoantibodies.Alloantibodies Produced by exposure to foreign red cell antigens which are non-self antigens but are of the same species. They react only with allogenic cells. Exposure occurs through pregnancy or transfusion. Examples include anti-K and anti-E. Autoantibodies Produced in an autoimmune process and directed against one's own red cell antigens. React with patient's own cells and all cells tested. Can possibly mask the presence of other significant antibodies. It is very important to make sure that no underlying significant antibodies are present if an autoantibody is suspected. A positive direct antiglobulin test (DAT) or auto control could indicate the presence of an autoantibody. Examples include cold auto (P or I) or warm auto (Rh specificity).

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Antinuclear Antibody Testing: Methods and Pattern Interpretation
Follow-up Testing

The ANA test is the initial screening assay used by clinicians to assess the likelihood that the patient has one of the SARDs. If the ANA is positive it is common for the clinician to request specific follow-up testing to see if the specific autoantibody(ies) that is/are causing the positive ANA can be identified. If the autoantibody(ies) can be identified this may add significantly to the diagnosis of the patient. The ANA pattern will give the clinician valuable insight into which follow-up testing is appropriate. For a speckled ANA pattern, follow-up testing for antibodies directed to the extractable nuclear antigens (ENA) is indicated. ENA testing is most frequently done by ELISA method. For a homogeneous ANA pattern, follow-up testing for antibodies to dsDNA is appropriate with most labs using a fluorescent slide method (CLIF), ELISA, or RIA.Some laboratories add a comment to the results suggesting appropriate follow-up testing. Using the previous example, Sample 12345: ANA Positive, Speckled, titer: 1:640; suggest follow-up ENA testing.

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References

American College of Rheumatology, Committee on Rheumatologic Care, Position Statement, Methodology of Testing for Antinuclear Antibodies; Feb, 2009. Available at http://www.rheumatology.org/search/search.asp accessed on June 16, 2010Anuradah V, Chopra A, Sturgess A, Edmonds J. Cost-effective screening method for antinuclear antibody detection. Asian Pacific League of Associations for Rheumatology. 2004(7):13-18.Arbuckle MR, McClain MT, Rubertone MV, et al. Development of autoantibodies before the clinical onset of systemic lupus erythematosus. N Engl J Med. Oct 16 2003;349(16):1526-1533.Bossuyt X, Frans J, Hendrickx A, Godefridis G, Westhovens R, Marien G. Detection of Anti-SSA Antibodies by Indirect Immunofluorescence. Clin Chem. 10 7 2004;50(12):2361-2369.Clinical and Laboratory Standards Institute (formerly NCCLS); Quality Assurance of Laboratory Tests for Autoantibodies to Nuclear Antigens: (1) Indirect Fluorescence Assay for Microscopy and (2) Microtiter Enzyme Immunoassay Methods; Approved Guidelines - Second Edition. CLSI I/LA2-A2. 2006;26(13).Fritzler MJ, Hanson C, Miller J, Eystathioy T. Specificity of autoantibodies to SS-A/Ro on a transfected and overexpressed human 60 kDa Ro autoantigen substrate. J.Clin.Lab.Anal. 2002;16:103-108.Fritzler MJ, Miller BJ. Detection of autoantibodies to SS-A/Ro by indirect immunofluorescence using a transfected and overexpressed human 60 kD Ro autoantigen in HEp-2 cells. J.Clin.Lab.Anal. 1995;9:218-224.Fritzler MJ, Wall W, Gohill J, Kinsella TD, Humbel RL. The Detection of Autoantibodies on HEp-2 Cells Using an Indirect Immunoperoxidase Kit (Colorzyme®). Diag Immunol. 1986;4:217-221. Keech CL, Howarth S, Coates T, Rischmueller M, McCluskey J, Gordon TP. Rapid and sensitive detection of anti-Ro (SS-A) antibodies by indirect immunofluorescence of 60kDa Ro HEp-2 transfectants. Pathology. 1996;28:54-57.Keech CL, McCluskey J, Gordon TP. Transfection and overexpression of the human 60-kDa Ro/SS-A autoantigen in HEp-2 cells. Clin.Immunol.Immunopathol. 1994;73:146-151.Kroshinsky D, Stone JH, Bloch DB, Sepehr A. Case records of the Massachusetts General Hospital. Case 5-2009. A 47-year-old woman with a rash and numbness and pain in the legs. N Engl J Med. Feb 12 2009;360(7):711-720. McCarty, G.A., Valencia, D.W., and Fritzler, M.J., Antinuclear Antibodies-Contempory Techniques and Clinical Application to Connective Tissue Disease. New York: Oxford University Press, Inc. 1984. Murray DL, Homburger HA, Horvat RT, Snyder MR, College of American Pathologists; S-C 2009: Antinuclear Antibody Screening Methods; CAP Surveys S-C Diagnostic Immunology;2009 Pollock W, Toh BH. Routine immunofluorescence detection of Ro/SS-A autoantibody using HEp-2 cells transfected with human 60 kDa Ro/SS-A. J.Clin.Pathol. 1999;52:684-687.Singer, M. and Berg, P., Genes & Genomes-A Changing Perspective. Mill Valley, CA: University Science Books. 1991.Sullivan KE. The complex Genetic Basis of Systemic Lupus Erythematosus, Reprint from 1999 and 2000; Lupus Foundation, Available at http://www.lupus.org/education/articles/geneticbasis.html Accessed June 16, 2010.Wallace DJ. New methods for antinuclear antibody testing: does it cut costs and corners without jeopardizing clinical reliability? Nat Clin Pract Rheumatol. Aug 2006;2(8):410-411.Willcocks LC, Carr EJ, Niederer HA, et al. A defunctioning polymorphism in FCGR2B is associated with protection against malaria but susceptibility to systemic lupus erythematosus. Proc Natl Acad Sci U S A. Apr 27 2010;107(17):7881-7885.

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Blood Banking Question Bank - Review Mode (no CE)
Which of the following antigen groups is closely related to the ABO system:View Page

Detecting and Evaluating Coagulation Inhibitors and Factor Deficiencies (retired 5/27/2014)
Introduction: Factor Deficiencies

A deficiency in one or more coagulation factor will also cause abnormalities in hemostasis. The image to the right depicts the coagulation cascade. Notice how one factor acts upon another to eventually form a stabilized fibrin clot, the end product of the coagulation cascade. Having an abnormally low level, or a complete lack, of a coagulation factor can cause the extrinsic, intrinsic, or common pathways to malfunction, resulting in dangerous hemorrhagic issues including spontaneous bleeding. Two of the most common factor deficiencies are factor VIII (hemophilia A) and factor IX (hemophilia B). Hemophilia A comes in two forms: congenital (inherited) or acquired. Congenital hemophilia A represents the condition where an individual is born defecient (to various degrees) of factor VIII. Acquired hemophilia A is a condition in which an individual spontaneously produced an autoantibody to factor VIII, leaving the body unable to use the factor VIII that may be present. Hemophilia B is an inherited condition where the individual has a mutation of the factor IX gene and is unable to produce adequate levels of this coagulation factor.In some cases, patients have multiple factor deficiencies that are secondary to a primary condition such as vitamin K deficiency, disseminated intravascular coagulation (DIC), and liver disease. With vitamin K deficiency, the liver is unable to produce the coagulation factors that are vitamin K-dependent. During liver disease, the liver may be unable to produce coagulation factors effectively. In DIC, the clotting processes are in overdrive and will consume the coagulation factors that are being produced, leading to low levels of circulating coagulation factors.

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