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CLS 3311 Advanced Clinical Immunohematology. Antihuman Globulin Follow-Up Lecture. Tubes or saline contaminated with excess silicone Dirty glassware Overcentrifugation Over reading. May cause non specific aggregation May cause non specific aggregation
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CLS 3311Advanced Clinical Immunohematology Antihuman Globulin Follow-Up Lecture
Tubes or saline contaminated with excess silicone Dirty glassware Overcentrifugation Over reading May cause non specific aggregation May cause non specific aggregation RBC’s tightly packed results in difficulty dispersing – can mistake for agglutination Just looking too close False Positive: IAT or DAT
False Positive: IAT or DAT Polyagglutinable RBC’s • Inherited exposure of T, Tn, Tk antigens, also known as ‘crypt antigens’ that agglutinate with virtually all patient serum. • Using contaminated anti-serum • Patient has infection with enzyme secreting bacteria, the enzymes strip sialic acid from cell surface exposing ‘crypt antigens’. • Chapter 22, Harmening
False PositiveDAT’s • Complement component C4 may bind to red cell membrane of clotted specimens at 4C and Room temperature • May even occur in CPDA-1 (donor anticoagulant) red blood cells • can be avoided by using anticoagulant that chelates Ca++ such as EDTA
False PositiveDAT’s • Blood collected from IV lines used to infuse 5 or 10% dextrose in H2O or volume expanders • dextrose causes cells to aggregate • Septicemia or bacterially contaminated sample • bacterial enzymes remove sialic acid from RBC’s causing exposure of T antigens
False Negative:IAT’s & DAT’s Associated with Washing • Cells NOT washed adequately • Inadequate resuspension of RBC’s between washes • Contamination of cells during washing • Interruption, leaving cells in saline for prolonged period - Antibody elutes off of red blood cell
False Negative:IAT’s & DAT’s Associated with AHG Reagent • Deterioration of AHG in storage • Failure to add AHG to test • Delay in adding AHG to washed cells
False Negative: DAT Improper centrifugation • Undercentrifugation: agglutination may be missed • Overcentrifugation: fragile agglutination may be broken up trying to pry cells off tube Too many RBC’s • Ag excess:suspension too heavy - difficult to read Too few RBC’S • Too many cells lost during Washing step • Patient RBC suspension too weak
False Negative: DAT Monoclonal gammopathies • IgG concentration so high even 4 washes don’t completely remove free IgG - neutralize anti-IgG Less than 200 IgG’s attached per RBC • IgG concentration so low there’s not enough to attach onto red cell membrane to detect their presence
False Negative:IAT RBC’S and/or serum stored improperly • Can cause loss of RBC antigen’s • Can cause reduction of antibody activity Presence of Antibody that does not react with monoclonal anti-IgG • Can only be detected with anti-C’ or may be IgA
False Negative:IAT • Improper incubation • Temperature not at 37 C (=/– 1oC) • Incubation time not adequate for agglutination to form • Incubation time so long, Antibodies begin to dissociate • Patient serum not added • Poor reading technique