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

▶

May 10, 2023 525 likes •627 views

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

SLIDE 1

Platelet Surface Associated IgG Assay using flow cytometry

PLT PSIgG anti-dog IgG-FITC emitted fluorescent light energy  = 530 nm 488 nm fluorescence intensity emitted light  binding sites

Argon lazer

Reagent = anti-dog IgG-FITC

SLIDE 2

PSAIgG test in healthy dogs

PSAIgG Cut off < 14% for normal dog samples that are  4 hour old < 23% for normal dog samples that are  24 hour old

Negative control Ab Fluorescence Fluorescence

SLIDE 3

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

SLIDE 4

English Cocker –Primary IMT

Before treatment After 10 days of Prednisone 39% Positive 14% Positive Size Size Fluorescence

SLIDE 5

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

SLIDE 6

Dog with Rocky Mountain Spotted Fever

Before treatment After 2 days of doxycycline

19% positive

SLIDE 7

Dog recovered with treatment

Cold IV fluids and doxycycline Responded to treatment by 48 hours

SLIDE 8

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.

SLIDE 9

Time course development

f thrombocytopenia

and PSAIgG in dogs infected with Babesia gibsoni

Wilkerson et al, 2001;30 Vet. Clin. Path.

5 10 15 20 25 30 35 40 45

Platelets X 103/ml

50 100 150 200 250 300 350 400 450 500 Uninfected Control Chronically infected (day -60) Infected Day 0 Reinfected Day 0 Splenetomized

5 10 15 20 25 30 35 40 45

Percent PSAIgG

10 20 30 40 50

Days post infection PSAIgG may be transient