CLS 3311 Advanced Clinical Immunohematology

1. CLS 3311Advanced Clinical Immunohematology Antihuman Globulin Follow-Up Lecture

2. 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

3. 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

4. False PositiveDAT’s • Complement component C4 may bind to red cell membrane of clotted specimens at 4C 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

5. 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

6. 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

7. 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

8. 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

9. 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

10. 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

11. 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