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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 3311 advanced clinical immunohematology

CLS 3311Advanced Clinical Immunohematology

Antihuman Globulin Follow-Up Lecture

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

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 dat3
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 positive dat s
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
false positive dat s5
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
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 s7
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
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 dat9
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
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 iat11
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