Stability of b2-transferrin and b-trace protein

Lyn Boscato Chemical Pathology, St Vincent’s Hospital, Sydney, Australia. [email protected];

Introduction and Aim Study 1: Effect of saliva on B2T

b2-transferrin (B2T) and b-trace protein (BTP) are useful markers for SUSPECTED SAMPLE PROBLEM the diagnosis of CSF leakage. A sample received for routine investigation of a suspected CSF Specimens received for analysis are often heavily contaminated with leak was negative for BTP but the transferrin isoform pattern other substances (eg blood, serous fluid, saliva, bacteria, mucus) and suggested CSF presence. Sample was an oral fluid collection so stored under non-ideal conditions (not frozen, large container, very small sample volume, on swabs). sialidase presence was suspected as the oral cavity can have a high bacterial load The aim of this study was to investigate the stability of B2T and BTP in 1 2 3 4 5 the presence of potential contaminants. 1 - CSF 4 - serum+ saliva STUDY 2 - CSF + saliva 5 - serum To determine if saliva contains sialidase 3 - saliva • • Saliva collected by passive drooling and microfuged to remove Figure 1. Transferrin isoforms detected following particulates. Methods IEF- western blotting for CSF and serum incubated • Equal volumes of saliva and serum or CSF were incubated with and without saliva. overnight at RT • Transferrin isoforms detected STUDIES  Investigation of the stability of B2T and BTP incubated in the presence of a. saliva as a source of sialidase (enzyme which removes sialic acid from glycoproteins. Normally arises from bacterial or viral Study 2: Stability of B2T and BTP sources). See Study 1

b. red cell haemolysate as a source of proteolytic enzymes 120 at room temperature (RT) and 4° for up to 1 month STUDY 110

 Investigation of the effect of storage of a small sample in a large • CSF specimens obtained for routine CSF analysis were used

container on the BTP level. Saliva was collected using a Salivette collection device BTP • 100 ANALYSES • Red cell haemolysate was prepared by freezing and thawing EDTA whole blood (plasma removed and replaced with saline) 90  BTP measured using the NLatex bTP on the Siemens BNProspec Saline • 0.7mL CSF and 0.3mL saliva/ haemolysate (Hb 1519mg/dL)/

 B2T detected by Western blotting following isoelectric focussing Recovery Saliva

saline (control) were incubated up to 1 month at RT or 4°. % 80  Protein measured using the U/CSF protein on the Roche Modular Haemosylate (sample diluted in saline due to small volume) • Aliquots were taken and stored frozen at -27° prior to batch analysis. 70  Drying of sample estimated from loss of sample weight 0 5 10 15 20 25 30 35 As there was no change in BTP between the zero time and last • Time (days) sample collected when stored at RT, samples for other times or 4’ were not tested. Figure 2. % of the zero time BTP level obtained after storage for various CSF specimens incubated with saline Results Summary (control n = 10), saliva (n = 5) or haemolysate (n = 2)

• Saliva contains sialidase which can result is a reduction in the more A B C D S H1 H2 H1 H2 sialylated forms of transferrin and a subsequent increase in less sialylated forms including asialotransferrin (B2T) (Figure 1) • Mean recovery of BTP was 94% for CSF (n=10), 98% for CSF incubated with saliva (n=5) and 94% for CSF with haemolysate 0 10 0 10 0 10 0 10 0 6h 6h 6h 10d 10d 0 1h 4h 1d 4d 1w 2w 4w (n=2) when stored at RT for at least 10 days (Figure 2). Figure 3. Transferrin isoforms for CSF Figure 4. Transferrin isoforms for Figure 5. Transferrin isoforms for CSF-F • The CSF B2T isoform pattern was not altered following 10 days specimens (A-D) stored for 0 or 10 days CSF –E incubated at RT with saliva incubated with saliva for various times incubation at RT with or without haemolysate (Figure 4&3) at RT. (S) or haemolysate (H1 &H2) for (w-weeks, d-days, h-hours) at RT • Presence of sialidase (saliva) resulted in an increase in the various times (d-days, h-hours) desialylated forms of transferrin after 0.5 days of storage at RT and then the complete disappearance of normal transferrin after 1 day (Figure 5 and 4). Study 3: Effect of small sample volume on BTP • Storage of small volumes of fluid in a large container can result in sample drying and an increase in total protein and BTP 2.5

concentration (Figure 6). The average %increase in BTP and protein LOW SAMPLE VOLUME SPECIMENS

respectively was for the 25mL samples - 80 and 60; 50mL- 30 and BTP Low sample volume (eg<100mL) specimens are often Protein 2 received for investigation of suspected CSF leakage in our 30. 100 and 200mL samples were not increased significantly. Concentration factor laboratory. They are normally sent a large container such 1.5 as a 50mL urine pot and the specimen is distributed in tiny droplets around the container. Obvious drying of the specimen is sometimes present. These are often serous Discussion and Conclusion 1 fluids and the BTP level above the upper limit for serum.

0.5 STUDY BTP and B2T in untreated CSF are stable for at least 10 days at RT.

Ratio (Test sample/Control) (Test Ratio To determine the effect on the BTP level of storage of small volume The proteolytic enzymes in haemolysate do not effect transferrin samples in a large container. 0 isoforms or the BTP level. 0 50 100 150 200 250 25-200mL of specimen (serous fluids not containing CSF; protein 12- The presence of sialidase in the specimen does not alter the BTP Sample volume (mL) 41g/L, BTP 0.3-1.04 mg/L, n=8) was placed in a urine jar which was concentration but can lead to a misleading interpretation for B2T. closed, shaken to distribute the sample around the jar and stored at RT for 3 days. CSF from the same samples was also stored in large In situations where sialidase contamination arising from bacterial or Figure 6. Ratio of the BTP and protein level of the test volume in a 0.5mL microfuge tube as control. viral sources invalidates theB2T interpretation, BTP measurement samples compared to control for small sample volumes stored Protein and BTP were measured on control, and sample retrieved remains valid. in a large container for 3 days at RT (Control – large volume from the container when sufficient remaining specimen was As the interpretation of BTP is dependant on a quantitative result, in- of the same sample stored in a small container). Concentration factor – refer to text available. Two 25uL samples dried completely. Theoretical vivo (eg small volume of fluid in ear canal) or in-vitro situations (eg concentration of the sample was estimated from the weight loss on small volume sample in a large container) that result in sample storage (1 / %weight loss) and labelled as concentration factor in concentration could lead to a misleading BTP result. Figure 6.

AACB Annual Scientific Meeting, Melbourne, 2012