Table 1.

Algorithm of study serum samples storage.

Storage Period	Group I	Procedure
24 (h)	RT; 4 °C	Examination of serum
48 (h)
120 (h)
Storage Period	Group II	Procedure	Group III	Cycle	Procedure
1 (M)	−20 °C; −80 °C	Thawing and examination of serum	−20°; −80 °C	T/F	Thawing, and re-freezing of serum
2 (M)				R1	Thawing, examination and re-freezing of serum
3 (M)				R2
4 (M)				R3
6 (M)				R4

RT—room temperature; T/F—thawing and re-freezing of the same samples; R1,2,3,4—cycles of thawing and re-freezing of the same samples; (h)—hour; (M)—month. Each patient’s serum sample was divided into 24 separated aliquots (3 portions were stored at RT, 3 at 4 °C, 5 at −20 °C, 5 at −80 °C, 4 at −20 °C/T/F, 4 at −80 °C/T/F) in which the determination of the studied parameters was examined only once. All of the patients’ samples subjected to cyclic temperature changes (aliquots for −20 °C and aliquots for −80 °C) were thawed; one sample stored at −20 °C and one stored at −80 °C were used to perform the analysis, the remaining samples were frozen again for further testing.