MATRIX AND STABILITY STUDY: PTH, INSULIN AND C-PEPTIDE

1. 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. Stability APACB 2004

2. 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. Stability APACB 2004

3. 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. Stability APACB 2004

4. 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. Stability APACB 2004

5. 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). Stability APACB 2004

6. PTH - Immulite 160 Serum RT 140 Serum 4°C 120 Heparin RT 100 Heparin 4°C 80 EDTA RT EDTA 4°C 60 40 20 0 0 20 60 40 Time (h) % of Serum Tube % Serum 100 Plasma 107 EDTA 117 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 Stability APACB 2004

7. 120 120 100 100 0 80 80 6 24 60 60 40 40 20 20 0 0 Serum Serum H+ Serum H++ Heparin Heparin H+ Heparin H++ Time(h) HAEMOLYSIS % 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 Stability APACB 2004

8. Insulin - Bayer Centaur 120 Serum RT Serum 4°C 100 Heparin RT 80 Heparin 4°C % of Serum EDTA RT 60 FlOx RT 40 Tube % Serum 100 Plasma 94 EDTA 97 FlOx 80 20 0 0 20 40 60 Time(h) 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 Stability APACB 2004

9. HAEMOLYSIS 0 6 24 Time(h) 120 120 100 100 80 80 % 60 60 40 40 20 20 0 0 Serum Serum H+ Serum H++ Heparin H+ Heparin H++ Heparin 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 • 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) Stability APACB 2004

10. C-peptide - Linco Serum RT Serum 4°C Heparin RT Heparin 4°C EDTA RT FlOx RT 120 100 80 % of Serum 60 40 Tube % Serum 100 Plasma 99 EDTA 96 FlOx 87 20 0 0 20 40 60 Time(h) 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 Stability APACB 2004

11. HAEMOLYSIS 120 120 100 100 80 80 0 6 60 60 24 40 40 20 20 0 0 Heparin Heparin H+ Heparin H++ Serum Serum H+ Serum H++ 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 • 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) Stability APACB 2004

12. C-peptide - Bayer Centaur 120 Serum RT Serum 4°C 100 Heparin RT 80 Heparin 4°C % of Serum EDTA RT 60 FlOx RT 40 Tube % Serum 100 Plasma 100 EDTA 88 FlOx 86 20 0 0 20 40 60 Time(h) 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 Stability APACB 2004

13. HAEMOLYSIS 120 120 100 100 0 80 80 6 60 60 24 40 40 20 20 0 0 Heparin Heparin H+ Time(h) % Serum Serum H+ Serum 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 • 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) Stability APACB 2004

14. 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). Stability APACB 2004

15. 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. Stability APACB 2004