1. Use of BVBlue ® to Confirm Sialidase Contamination of Samples for ß2transferrin Determination. L Boscato Department of Chemical Pathology St Vincent’s Hospital, Sydney

2. 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

3. Detection of Transferrin Isoforms Isoelectric focussing Western blotting Immunodetection - peroxidase antibody 0 4 SerumCSFSample dilutionsCSF + - Sialic Acid Residues

4. 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

5. Detection of Interference SerumCSFSample 1/100 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

6. 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.

7. Studies Does it work? Incubation conditions - time, sample volume Variability in response Sensitivity

8. 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

9. Incubation Conditions 0 0.2 0.4 0.6 0.8 1 1.2 1.4 050100150 0 0.5 1 1.5 2 2.5 3 3.5 0100200300400 TimeSample volume Time (mins)Volume (uL) OD 590nm

10. Sialidase negative samples 0 0.5 0.10 0.15 0.20 0.25 Normal CSF CSF+veCSF-veCSF+ve +serum OD 590nm

11. Sialidase positive samples OD 590nm CSFCSF+veCSF-veCSF+ve +serum Sialidase 0 0.50 1.00 1.50 2.00 2.50 3.00

12. Purified Sialidase Detection Sialidase (uU) OD 590nm Gel Method 0 0.5 1.0 1.5 2 050100150200250 ++ --

13. 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

14. 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.

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