1. Stability APACB 20041 MATRIX AND STABILITY STUDY: PTH, INSULIN AND C-PEPTIDE Boscato L, Bayoun R and Jones GRD Chemical Pathology, St Vincent’s Hospital, Darlinghurst, NSW 2010.

2. Stability APACB 20042 Introduction We are frequently requested to measure analytes in specimen types other than those recommended or in samples not stored under ideal conditions Information on sample type and stability supplied by the assay manufacturer is often limited and conservative. It can therefore be difficult to determine if measurement of analyte in ‘non-ideal’ samples is valid. This could result in unnecessary recollection. More detailed information of the effect of sample type and storage condition on the analyte level is required.

3. Stability APACB 20043 Aim of Study Parathyroid hormone(PTH) and c-peptide are generally considered to be ‘non-robust’ analytes for which sample type and storage conditions are important Information regarding insulin stability varies widely between kit inserts from different manufacturers We investigate the effect of sample matrix and storage conditions on the measured levels of parathyroid hormone (PTH), c-peptide and insulin.

4. Stability APACB 20044 Methods Blood was collected from 4 healthy volunteers (non- fasting) and three renal failure patients (for PTH) into serum (SSTII), heparin (LH PST), fluoride oxalate (FlOx) and EDTA tubes. All tubes were BD Vacutainer brand Samples were placed on ice within 15 minutes of collection. Following separation (4°C) samples were stored at 4° or room temperature (RT) for up to 48 hours and then frozen at –27° prior to batch analysis. Samples were assayed within 2 weeks after collection.

5. Stability APACB 20045 Methods The effect of haemolysis was investigated using specimens with added haemolysate Haemolysate was prepared by freezing and thawing heparin whole blood (plasma removed and replaced with distilled water) Haemoglobin at 200mg/dL (H+) and 800mg/dL (H++) were tested The analytes measured were: PTH (DPC Immulite) insulin (Bayer Centaur), c-peptide (Bayer Centaur and Linco RIA).

6. Stability APACB 20046 PTH - Immulite Tube % Serum 100 Plasma 107 EDTA 117 Serum RT Serum 4 °C Heparin RT Heparin 4 °C EDTA RT EDTA 4 °C % of Serum Fig 1. Mean PTH levels (% of the 0 serum value)obtained for a number of sample types after storage for various times at RT or 4 °C 0 20 40 60 80 100 120 140 160 0 20 40 60 Time (h)

7. Stability APACB 20047 0 20 40 60 80 100 120 SerumSerum H+Serum H++ 0624 Time(h) Fig 2. Effect of added haemolysate (H+, H++ ) on the measured PTH(%of zero) levels of serum and heparin plasma stored at RT for 0, 6 and 24 hours. PTH Heparin plasma - 107% of serum value EDTA- 117% of serum values and level increases with time at RT Serum RT/4° and heparin 4° - stable up to 48 hours Serum and plasma PTH levels are reduced by haemolysis (dose dependent) Serum PTH more affected but the degree of loss independent of storage time HAEMOLYSIS % HeparinHeparin H+Heparin H++ 0 20 40 60 80 100 120

8. Stability APACB 20048 Insulin - Bayer Centaur 0 20 40 60 80 100 120 0204060 Tube % Serum 100 Plasma 94 EDTA 97 FlOx 80 Serum RT Serum 4 °C Heparin RT Heparin 4 °C EDTA RT FlOx RT % of Serum Fig 3. Mean insulin levels (% of the 0 serum value) obtained for a number of sample types after storage for various times at RT or 4 ° C Time(h)

9. Stability APACB 20049 0 20 40 60 80 100 120 SerumSerum H+Serum H++ 0 20 40 60 80 100 120 Heparin Heparin H+Heparin H++ 0624 Time(h) Fig 4. Effect of added haemolysate (H+, H++ ) on the measured insulin (% of zero) level of serum and heparin plasma stored at RT for 0, 6 and 24 hours HAEMOLYSIS Insulin EDTA insulin levels - similar to serum - 97%(Fig 3) Fluoride oxalate - 80% of serum levels, heparin Plasma - 94% of serum Significant loss of insulin at RT particularly for heparin (20% loss at 6 h) Insulin is highly susceptible to degradation due to haemolysis with significant loss even at zero time (heparin 46%, serum 10%). Higher loss with heparin plasma (Fig 4) %

10. Stability APACB 200410 C-peptide - Linco 0 20 40 60 80 100 120 0204060 Tube % Serum 100 Plasma 99 EDTA 96 FlOx 87 Serum RT Serum 4 °C Heparin RT Heparin 4 °C EDTA RT FlOx RT % of Serum Fig 5. Mean c-peptide levels (% of the 0 serum value) obtained for a number of sample types after storage for various times at RT or 4 ° C Time(h)

11. Stability APACB 200411 0 20 40 60 80 100 120 SerumSerum H+Serum H++ 0 20 40 60 80 100 120 HeparinHeparin H+Heparin H++ 0624 Time(h) Fig 6. Effect of added haemolysate (H+, H++ ) on the measured c-peptide level (% of zero) of serum and heparin plasma stored at RT for 0, 6 and 24 hours HAEMOLYSIS C-peptide - Linco Serum and heparin plasma - values identical, EDTA 96% of serum (Fig 5) Fluoride oxalate - 87% of serum value (Fig 5) C-peptide in serum and heparin plasma stable at RT up to 48 hours (Fig 5) Haemolysis reduces c-peptide levels in serum and heparin plasma particularly following storage at RT (Fig 6) %

12. Stability APACB 200412 C-peptide - Bayer Centaur 0204060 0 20 40 60 80 100 120 Tube % Serum 100 Plasma 100 EDTA 88 FlOx 86 Serum RT Serum 4 °C Heparin RT Heparin 4 °C EDTA RT FlOx RT % of Serum Fig 7. Mean c-peptide levels (% of the 0 serum value)obtained for a number of sample types after storage for various times at RT or 4 ° C Time(h)

13. Stability APACB 200413 0 20 40 60 80 100 120 SerumSerum H+Serum H++ 0 20 40 60 80 100 120 HeparinHeparin H+ 0624 Time(h) Fig 8. Effect of added haemolysate (H+, H++ ) on the measured c-peptide level (% of zero) of serum and heparin plasma stored at RT for 0, 6 and 24 hours HAEMOLYSIS C-peptide - Bayer Centaur Serum and heparin plasma - c-peptide levels identical (Fig 7) EDTA- 88% of serum value, Fluoride oxalate - 86% of serum value (Fig 7) Serum and heparin plasma stable at RT up to 48 hours (Fig 7) Haemolysis reduced c-peptide levels in serum (20% reduction with 200mg/dL). The loss is increased with storage time and amount of haemolysate. C-peptide in heparin plasma is less affected (Fig 8) %

14. Stability APACB 200414 Results Summary Of the three analytes tested the greatest decrease in analyte level was noted for insulin when samples were stored at room temperature (RT) with heparin plasma losing 20% immunoreactivity by 6 hours and serum 20% at 24 hours. Insulin was also found to be highly susceptible to interference due to haemolysis. Fluoride oxalate was found to be unsuitable for insulin and c-peptide. In contrast to the information supplied by Bayer and Linco c-peptide was stable in serum and heparin plasma stored at RT for up to 48 hours. C-peptide displays similar stability in the Linco and Bayer assays. PTH was stable in serum at RT for 48 hours in contrast to the kit insert information ( 8 hours at 2-8°C).

15. Stability APACB 200415 Conclusions Results indicate that PTH and c-peptide are more stable than current understanding suggests. Loss of immunoreactivity of these analytes may however occur during longer term frozen storage. Insulin levels are markedly affected by the storage temperature and degree of haemolysis. Information on sample stability/specimen type should reduce the need for recollection when collection or processing instructions have not been followed and for ‘add-on’ tests where the sample available is not the preferred specimen.