HIT Heparin Induced Thrombocytopenia Testing by ELISA

1
HIT (Heparin Induced Thrombocytopenia) Testing by
ELISA

• Immune mediated Heparin Induced Thrombocytopenia
  (HIT) generates antibodies to Platelet Factor 4
  (PF4) complexed with heparin. Two currently
  available solid phase enzyme linked immunoassays
  (ELISA) utilize either recombinant PF4 complexed
  with unfractionated heparin (Diagnostica Stago)
  or PF4 complexed with polyvinylsulfonate by GTI.
  In either assay, the complexing of positive PF4
  to negatively charged heparin causes a cryptic
  autoantigen within PF4 to be recognized by HIT
  antibodies. ELISA assays are antigenic, not
  functional, hence they are not able to show
  platelet activation caused by the antibody.
• Warkentin Greinacher, HIT. Marcel Dekker, 2000
• Walenga, JM et al. Semin Thromb Hemost,199925
  (Suppl 1)43-9
• Izban KF, et al. Semin Thromb Hemost,199925
  (Suppl 1)51-6

Lab Pointer
Platelet Factor 4
Revised mmiiii
www.CLOT-ED.com
M Ledford-Kraemer
2
HIT (Heparin Induced Thrombocytopenia) Testing by
ELISA
Lab Pointer
Revised mmiiii
www.CLOT-ED.com
M Ledford-Kraemer
3
HIT (Heparin Induced Thrombocytopenia) Testing by
ELISA

• Assay Considerations
• ELISA assays are screening assays
• False positives observed pregnancy, elderly,
  diabetics, samples containing immune complexes, 4
  days post cardiac surgery
• False negatives due to low titer, low avidity
  antibodies
• HIT pathogenic antibodies are generally IgG both
  commercial assays may be overtly sensitive since
  they detect all three antibody classes
• Both commercial assays validated only for
  unfractionated heparin
• It can not be concluded that any thrombocytopenic
  patient who has detectable PF4-reactive
  antibodies automatically has HIT. The clinical
  context must be taken into account.

Lab Pointer

• Technical Considerations
• Assay is affected by variations in temperature,
  exposure of substrate to stray light, or
  bacterial contamination of reagents
• Assay sensitivity can be increased by spiking
  sample buffer with high concentrations (100U/ml)
  of heparin
• If PF4 antibodies are present, results will be
  negative with high heparin concentration since
  antibody will be masked

Revised mmiiii
www.CLOT-ED.com
M Ledford-Kraemer