Cliff Threlkeld, D.O.Central Florida Research

A New Diagnostic Test for Lyme Disease

Introduction – using flow cytometry to detect Borrelia burgdorferi (Bb)Basic principles of flow cytometryApplication to Lyme disease testingComparison of flow cytometry to other methods

of Lyme disease testingAvailability of flow cytometry Lyme disease

testing

A New Diagnostic Test for Lyme Disease

Basics of flow cytometryTypical flow cytometer - power, mechanical

and detection, and computer systemTypical setupFlow cellHow are events detected?How antibodies work

Typical Flow CytometerTypical Flow Cytometer

Computer SystemDetector &Mechanical

Power

Cytometer

Power

PC & Printer

Specimen Tube

Injector Tip

FluorescenceFluorescencesignalssignals

Focused laserFocused laserbeambeam

•Sheath• fluid

Flow CytometryFlow Cytometry

PMT FITC

PMT PE

PMT ECD

PMT PC5

DichroicFilters

Laser

1

2

3

4

Flow cell

Bandpass Filters

319

ZAP70

5

FS

SS

How do Abs work?

A New Diagnostic Test for Lyme DiseaseApplication of flow cytometry to Lyme disease

testingTraditional two-test protocol antibody testing - ELISA

and Western Blot (WB)Bacterial detection methods – Culture and polymerase

chain reaction (PCR) – Flow cytometry underutilizedUsing flow cytometry to detect Borrelia burgdorferi in

patient blood Antibody with fluorescein label (goat, KPL) added to aliquot

of patient blood (EDTA) Incubation for 1 hour at 37 degrees C Analyze by FC – Bb are bright fluorescent events

A New Diagnostic Test for Lyme Disease

Comparison of FC to WB and PCRPhase 1 study compared FC with WB66 patients with clinical diagnosis of LD

5 positive by WB, 4 of those positive by FC An additional 28 patients positive by FC

A New Diagnostic Test for Lyme Disease

Comparison of FC to PCRPhase 2 compared FC to PCR in 42 patients

with a clinical diagnosis of LD16 patients positive by FC and 18 positive by

PCR 8 patients positive by PCR and FC Gene sequencing performed on 14 PCR positive

patients (5 FC positive) 8 Bb sensu stricto (4 FC) 7 Bb sensu lato (new subgroup, 2 by FC)

A New Diagnostic Test for Lyme Disease

Sensitivity and specificity of FC and WB comparedSensitivity FC = 66% Specificity = 92%Sensitivity WB = 52% Specificity = 100%

A New Diagnostic Test for Lyme Disease

Sensitivity and specificity of FC and PCR comparedSensitivity FC = 62% Specificity = 100%Sensitivity PCR = 64% Specificity = 100%

A New Diagnostic Test for Lyme Disease

Availability of flow cytotometry LD testingCentral Florida Research, Winter Haven,

Florida Web site – centralfloridaresearch.com

A New Diagnostic Test for Lyme Disease

Future applicationsTesting for multiple tick-borne disease

pathogens simultaneouslyAdd antibodies to European Borrelia speciesLook for Bb antigens within monocytes