Use of BVBlue® to Confirm Sialidase Contamination of Samples for ß2transferrin Determination.

L Boscato Department of Chemical Pathology

St Vincent’s Hospital, Sydney

Introduction

• Multiple isforms of transferrin exist which vary in number of sialic acid residues.

• CSF is distinguished from other fluids by – total transferrin concentration– increased proportion of transferrin lacking sialic

acid [ß2transferrin (ß2T), asialotransferrin, tau protein]

• Detection of ß2T in discharges from the ear, nose or wounds is a useful tool in the investigation of suspected CSF leakage

Detection of Transferrin Isoforms

• Isoelectric focussing• Western blotting• Immunodetection - peroxidase antibody

0

4

Serum CSF Sample dilutions CSF

+

-

SialicAcid

Residues

• Spurious elevation of ß2T resulting in incorrect interpretation of CSF presence could lead to inappropriate patient management

• False elevation of ß2T can arise from bacterial sialidase activity in the sample

The Problem

Detection of Interference

Serum CSFSample1/100

Serum+Fluid

GEL STUDY: Incubation of serum with fluid - monitor products• time consuming• transferrin forms in fluid can mask products

Sample

0

4

0

4

Study

Aim of Study

• BVBlue® is a point of care enzyme activity test for detection of bacterial sialidase activity in vaginal swabs and is used for diagnosis of bacterial vaginosis.

• The aim of the study was to assess the potential of the BVBlue® test for the confirmation of sialidase activity in fluids thought to contain CSF.

Studies

• Does it work?• Incubation conditions - time, sample volume• Variability in response• Sensitivity

BVBlue®

• incubation of sample(37°C) with chromogenic substrate

• addition of colour developer- blue colour

• OD at 590nm superior to visual assessment for samples with lower levels of activity

• negative control required

Incubation Conditions

0

0.2

0.4

0.6

0.8

1

1.2

1.4

0 50 100 1500

0.5

1

1.5

2

2.5

3

3.5

0 100 200 300 400

Time Sample volume

Time (mins) Volume (uL)

OD

590

nm

Sialidase negative samples

0

0.5

0.10

0.15

0.20

0.25

Normal CSF

CSF+ve CSF-ve CSF+ve+serum

OD

590

nm

Sialidase positive samplesO

D 5

90n

m

CSF CSF+ve CSF-ve CSF+ve+serum

Sialidase

0

0.50

1.00

1.50

2.00

2.50

3.00

Purified Sialidase Detection

Sialidase (uU)

OD 590nm

GelMethod

0

0.5

1.0

1.5

2

0 50 100 150 200 250

+ +- -

Summary

• BV Blue® is able to detect sialidase contamination in samples

• Sensitivity can be enhanced – with spectrophotometric reading– by increasing sample volume and incubation time

• BVBlue® more sensitive than current gel method for detecting interference

Conclusions

• BVBlue ® is a viable alternative to the current technique for detecting sialidase activity

• Ease of use, speed and sensitivity make the BVBlue® test suitable for routine screening of unusual specimens.

BVBlue®

These powerpoint slides will be available on www.sydpath.stvincents.com.au after the conference.