CLS 3311 Advanced Clinical Immunohematology

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CLS 3311Advanced Clinical Immunohematology

• Antihuman Globulin Follow-Up Lecture

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False Positive IAT or DAT

• Tubes or saline contaminated with excess silicone
  
• Dirty glassware
• Overcentrifugation
• Over reading

• May cause non specific aggregation
• May cause non specific aggregation
• RBCs tightly packed results in difficulty
  dispersing can mistake for agglutination
• Just looking too close

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False Positive IAT or DAT

• Polyagglutinable RBCs
• 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

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False PositiveDATs

• 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

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False PositiveDATs

• 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 RBCs
  causing exposure of T antigens

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False Negative IATs DATs

• Associated with Washing
• Cells NOT washed adequately
• Inadequate resuspension of RBCs between washes
• Contamination of cells during washing
• Interruption, leaving cells in saline for
  prolonged period - Antibody elutes off of red
  blood cell

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False Negative IATs DATs

• Associated with AHG Reagent
• Deterioration of AHG in storage
• Failure to add AHG to test
• Delay in adding AHG to washed cells

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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 RBCs
• Ag excess suspension too heavy - difficult to
  read
• Too few RBCS
• Too many cells lost during Washing step
• Patient RBC suspension too weak

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False Negative DAT

• Monoclonal gammopathies
• IgG concentration so high even 4 washes dont
  completely remove free IgG - neutralize anti-IgG
• Less than 200 IgGs attached per RBC
• IgG concentration so low theres not enough to
  attach onto red cell membrane to detect their
  presence

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False Negative IAT

• RBCS and/or serum stored improperly
• Can cause loss of RBC antigens
• 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

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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