Transcript A SIMPLE PROTOCOL FOR IDENTIFICATION OF HETEROPHILIC

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
Sialic
Acid
4 Residues
0
+
Serum
CSF
Sample dilutions CSF
The Problem
• 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
Detection of Interference
GEL STUDY: Incubation of serum with fluid - monitor products
• time consuming
• transferrin forms in fluid can mask products
Study
0
0
4
4
Serum
+Fluid
Serum Sample Sample CSF
1/100
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
Time
Sample volume
3.5
1.4
3
1.2
2.5
1
2
0.8
1.5
0.6
1
0.4
0.5
0.2
0
0
0
100
200
Time (mins)
300
400
0
50
100
Volume (uL)
150
Sialidase negative samples
0.25
OD 590nm
0.20
0.15
0.10
0.5
0
Normal
CSF
CSF+ve
CSF-ve
CSF+ve
+serum
Sialidase positive samples
3.00
OD 590nm
2.50
2.00
1.50
1.00
0.50
0
CSF
CSF+ve
CSF-ve
CSF+ve
+serum
Sialidase
Purified Sialidase Detection
Gel
Method
--
+
+
2
1.5
OD 590nm
1.0
0.5
0
0
50
100
150
Sialidase (uU)
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.