Platelet Surface Associated IgG Assay using flow cytometry emitted - PowerPoint PPT Presentation

Platelet Surface Associated IgG Assay using flow cytometry emitted fluorescent light energy Argon lazer anti-dog IgG-FITC  = 530 nm 488 nm emitted light  binding sites PLT PSIgG fluorescence intensity Reagent = anti-dog IgG-FITC

PSAIgG test in healthy dogs Negative control Ab Fluorescence Fluorescence PSAIgG Cut off < 14% for normal dog samples that are  4 hour old < 23% for normal dog samples that are  24 hour old

Case Example of Primary immune-mediated thrombocytopenia (IMT) 11 year old female spayed English Cocker  episodic thrombocytopenia responsive to corticosteroid therapy  1,000/  l  negative titers to tick borne diseases • RMSF, Lyme’s, Erhlichia  normal coagulation profiles  Mailed in sample

English Cocker –Primary IMT 39% Positive Size Before treatment 14% Positive Size After 10 days of Prednisone Fluorescence

Case Example of Secondary IMT 6 yr old male Labrador cross Admitted to KSU (In house case)  Fever (106 F)  Mild anemia (HCT = 30%)  64,000/  l platelets  Ataxia  Acute titer to RMSF 1:128  Convalescent titer to RMSF 1:2048

Dog with Rocky Mountain Spotted Fever Before treatment 19% positive After 2 days of doxycycline

Dog recovered with treatment Cold IV fluids and doxycycline Responded to treatment by 48 hours

Sensitivity/Specificity of PSAIgG  Sensitivity 87.5% (21/24 true positive) • 12.5% (3/24) false negatives – Causes of false negatives » Elution of antibody » Antibody against megakaryocytes or cytokines » Removal of antibody coated cells by the spleen  Specificity 100% • 9/9 nonimmune cases – Myelophthisis due to lymphosarcoma – Neoplasia (sequestration or consumption?) Wilkerson, Meinkoth, Harkin, Kocan, et al, 2001;30 Vet. Clin. Path.

Uninfected Control 500 Chronically infected (day -60) Infected Day 0 450 Reinfected Day 0 400 Splenetomized Platelets X 103/ml 350 300 250 Time course 200 150 development 100 of thrombocytopenia 50 0 and PSAIgG 0 5 10 15 20 25 30 35 40 45 50 PSAIgG may be transient in dogs infected 40 with Babesia gibsoni Percent PSAIgG 30 20 10 0 0 5 10 15 20 25 30 35 40 45 50 Days post infection Wilkerson et al, 2001;30 Vet. Clin. Path.