ORIGINAL ARTICLE Standardization of Cystatin C: Development of primary and secondary reference preparations S. Blirup-Jensen 1 *, A. Grubb 1 , V. Lindstro ¨m 1 , C. Schmidt 2 and H. Althaus 3 1 Department of Clinical Chemistry, University Hospital, Lund, Sweden; 2 Global Clinical Immunochemistry and OEM, Dako A/S, Glostrup, Denmark; 3 Dade Behring Marburg GmbH, a Siemens Company, Marburg, Germany A Primary Reference Preparation has been produced using pure, recombinant, Cystatin C in a solvent of 0.1 mol/ L KCl. Dry mass determination of the Primary Reference Preparation resulted in a Cystatin C concentration of 5.20 g/L. Agarose-electrophoresis and SDS-electrophoresis, as well as N-terminal sequencing, verified the purity, homogeneity and identity of Cystatin C in the Primary Reference Preparation. For the Secondary Reference Preparation, a serum pool was collected and stabilized. A pilot batch was made to verify the selected procedure and spiking with the pure, recombinant Cystatin C. The final Secondary Reference Preparation is now produced (4468 vials) and ready for value assignment and further characterization. Keywords: Cystatin C; dry mass determination; Primary Reference Preparation; Secondary Reference Preparation; standardization of Cystatin C Introduction Serum Cystatin C has been shown to be an excellent marker for glomerular filtration rate [1,2]. However, in the clinical routine using different methods, results vary owing to a lack of standardization. The goal of the IFCC Working Group on Standardization of Cystatin C is to produce and characterize both a primary and a secondary reference preparation for Cystatin C. Primary Reference Preparation Recombinant human Cystatin C was produced by expression in E. coli according to [3]. Cystatin C was purified from the cell extract using dialysis, anion exchange and cation exchange chromatography and gelfiltration. The purified Cystatin C was filled into bottles and lyophilized. The lyophilized Cystatin C was reconstituted using 0.1 mol/L KCl, then dialyzed against 0.1 mol/L KCl for 4 days with repeated replacements of the solvent; finally, it was adjusted to a concentration of approximately 5.2 g/L measured by refractometry. This solution was aliquoted in vials of 0.2 mL and labelled (see Figure 1): Cystatin C – Primary Reference Preparation IFCC Working Group. Lot 11082006 – Store at 280 ˚ C. Dry mass determination Dry mass determination was started immediately and performed according to [4]. All weighing was carried out on an analytical balance with five decimals. Pyrex glass vials with fitted lids were filled with 3 mL of either protein solution or solvent. The sequence of the weighing vials (Empty – Protein Solution – Solvent) was repeated four times. Drying took place in an oven at 90 ˚ C with a slight vacuum of 27 kPa over 7 days. Figure 2 shows the weight of the protein solution and the solvent as recorded over the 7 days. The concentra- tion of the Primary Reference Preparation for Cystatin C was measured to be: C p 55.197 ¡0.0078 g/L with a CV of 0.15 % and an uncertainty (k51) of 0.088 g/L. Agarose screen electrophoresis In order to characterize the Primary Reference Preparation, agarose screen electrophoresis was performed according to [5]. Figure 3 shows that Cystatin C is monomeric, pure, and has the expected mobility. SDS-PAGE Likewise, SDS polyacrylamide gel electrophoresis of the Primary Reference Preparation was performed according to [6]. Figure 4 shows that Cystatin C is monomeric, pure, and has the expected molecular mass compared to a marker. *Correspondence. S. Blirup-Jensen, Department of Clinical Chemistry, University Hospital, Lund, Sweden. Email: Soren.Blirup@med.lu.se The Scandinavian Journal of Clinical & Laboratory Investigation, Vol. 68, No. S241, June 2008, 67–70 ISSN 0036-5513 print/ISSN 1502-7686 online # 2008 Informa UK Ltd (Informa Healthcare, Taylor & Francis AS). DOI: 10.1080/00365510802150067 http://www.informaworld.com