Effect of Freeze/Thaw Cycles on Several Biomarkers in Urine from Patients with Kidney Disease

Abstract

Urine samples were collected from eleven randomly selected patients with kidney disease, including diabetic nephropathy, chronic nephritis, and nephritic syndrome. Urine samples were treated with one of four protocols for freezing and thawing: freeze directly and thaw directly; freeze directly and thaw by temperature gradient; freeze by temperature gradient and thaw directly; and freeze by temperature gradient and thaw by temperature gradient. After one to six freeze/thaw cycles at −20°C or −80°C, different biomarkers showed differential stabilities. The concentrations of total protein, calcium, and potassium did not change significantly after five freeze/thaw cycles at either −20°C or −80°C. Albumin could only sustain three freeze/thaw cycles at −20°C before it started to degrade. We recommend that urine be stored at −80°C as albumin and the organic ions could sustain five and six freeze/thaw cycles, respectively, using the simple “direct freeze and direct thaw” protocol. Furthermore, in most cases, gradient freeze/thaw cycles are not necessary for urine sample storage.

Introduction

Urine is easily and noninvasively collected, and is thus a good target for detecting biomarkers for the risk assessment, diagnosis, management, and treatment of various diseases. “Omics” research is leading to the discovery of an increasing number of biomarkers and is an important factor in the development of the field of personalized medicine. However, high-quality biospecimens are essential for the discovery of biomarkers with high specificity and sensitivity.¹

The stability of urine after cycles of freezing and thawing is very important to its quality. The ISBER Best Practices for Repositories indicates that the rate and method of freezing/thawing specimens can have serious effects on the viability of cells.² However, to our knowledge, different freezing/thawing methods have not been tested on urine. Previous studies have shown that urine forms precipitates during freeze/thaw cycles that depletes both calcium and proteins during storage at −20°C for 12 hours.³ The stability of albumin after freezing and thawing is controversial, with some reports indicating it does not have an effect, and some indicating that it causes a significant decrease in albumin content.⁴ The stabilities of different proteins after freezing and thawing can vary greatly.^5,6

The Metabolic Diseases Biobank in the Sixth People's Hospital in Shanghai has samples from more than 100,000 individuals, and fresh serum and urine are collected every day. The purpose of this study was to verify the stability of some representative biomarkers, including total protein, albumin, and ions, in the urine of people with kidney disease, after freezing and thawing.

Methods

Samples procedures

The study was approved by the Institutional Review Board of Shanghai Jiao Tong University Affiliated Sixth People's Hospital in accordance with the principles of the Helsinki Declaration. Written informed consent was obtained from each participant.

Urine samples (24 hours) were collected from eleven randomly selected patients with kidney disease. The samples were centrifuged at 4000 rpm for 5 min to remove precipitates and then assayed for baseline levels of different biomarkers. Each specimen was divided into two portions, one for storage at −20°C and the other for storage at −80°C. Each portion was divided into four aliquots for treatment by four different protocols: 1. Freeze directly and thaw directly (DFDT); 2. Freeze directly and thaw by temperature gradient (DFGT); 3. Freeze by temperature gradient and thaw directly (GFDT); 4. Freeze by temperature gradient and thaw by temperature gradient (GFGT). Each of the aliquots was further subdivided into six equal aliquots for analysis after one to six freeze/thaw cycles.

Quantitative procedures

The concentrations of albumin were determined by immunonephelometry using the BN II System (Siemens). Total protein was determined using a colorimetric method (Hitachi). Ions were determined using an ion-selective electrode and an electrolyte analyzer (Hitachi).

Statistical analysis

The Mann-Whitney U test was used to compare sets of analyses. Differences between sets of analyses resulting in probability values of less than 0.05 were considered significant.

Results

Table 1 shows the mean concentrations of total protein, albumin, calcium, and potassium from the urine of 11 patients at baseline (fresh urine) and after one to six freeze/thaw cycles with storage at both −20°C and −80°C. The DFGT and GFDT protocols gave very similar results for all analytes in almost all cases when compared to the DFDT and GFGT protocols, respectively, and thus the data are not shown. Freezing and thawing of the urine specimens up to six times with storage at −20°C and −80°C resulted in a significant decrease in total protein and albumin concentrations, regardless of which freeze/thaw protocol was used. Interestingly, the concentration of total protein did not show a significant difference after five freeze/thaw cycles using the DFDT protocol at either −20°C or −80°C. The albumin concentrations did not show a significant difference after five freeze/thaw cycles if the samples were treated using the DFDT protocol at −80°C. The potassium levels were stable for all six freeze/thaw cycles using the DFGT and GFGT protocols except when stored at −20°C. The calcium levels were stable for five and six freeze/thaw cycles at −20°C and −80°C, respectively. A similar result is observed for other ions such as sodium, chloride, and phosphorous (data not shown), which are stable for up to six freeze/thaw cycles at −80°C. The DFDT protocol at −80°C is the most successful for all four analytes (Fig. 1).

FIG. 1.

Concentrations (mean±standard deviation) of four biomarkers measured from urine of 11 kidney patients after one to six freeze/thaw cycles at −80°C using the DFDT protocol. BL, baseline; DFDT, freeze directly and thaw directly; X axis, number of freeze/thaw cycles.

Table 1.

Mean Concentrations of Total Protein, Albumin, Calcium, and Potassium in Urine of 11 Patients with Kidney Disease after 1–6 Freeze/Thaw Cycles, with Storage at −20°C (A) and −80°C (B) ¹

Number freeze/thaw cycles
(A) >−20°C	Freeze/thaw categories ²	Baseline ³	1	2	3	4	5	6
Total protein (g/L)	DFDT	2.48±1.51	2.33±1.49	1.45±0.98	1.18±0.86	1.32±0.97	1.16±1.02	0.75±0.58^*
	GFGT	2.48±1.51	2.05±1.27	1.64±0.97	1.34±0.92	1.26±0.97	1.66±1.44	0.90±1.04^*
Albumin(g/L)	DFDT	1.67±1.00	1.68±0.94	1.21±0.76	1.20±0.73	0.65±0.38^*	0.60±0.45^*	0.73±0.66^*
	GFGT	1.67±1.00	1.69±1.10	1.34±0.72	1.28±0.95	0.75±0.49^*	0.62±0.30^*	0.70±0.71^*
Ca(umol/L)	DFDT	1.64±1.52	1.59±1.71	1.14±1.18	0.99±1.14	1.13±1.10	1.03±0.87	0.69±0.60^*
	GFGT	1.64±1.52	1.37±1.38	1.13±1.11	1.25±1.40	1.10±0.95	1.30±1.12	0.75±0.86^*
K(mmol/L)	DFDT	18.81±9.26	18.33±10.94	14.95±9.05	13.53±8.25	15.89±9.87	16.54±9.38	12.83±7.35
	GFDT	18.81±9.26	16.61±9.08	15.84±8.38	16.18±9.47	16.62±9.72	20.51±10.95	10.71±7.47^*

(B) −80°C	Freeze/thaw categories ²	Baseline ³	1	2	3	4	5	6
Total protein (g/L)	DFDT	2.48±1.51	1.83±1.37	1.77±1.23	1.32±1.20	1.49±1.18	1.01±0.92	0.96±1.10^*
	GFGT	2.48±1.51	1.87±1.34	2.09±1.84	1.24±1.05	0.81±0.87^*	0.59±0.85^*	0.83±0.58^*
Albumin(g/L)	DFDT	1.67±1.00	1.60±1.10	1.41±0.97	1.60±1.08	1.35±1.17	1.46±1.95	0.59±0.39^*
	GFGT	1.67±1.00	1.57±0.90	1.08±0.63	1.34±1.08	0.84±0.63	0.96±0.78	0.60±0.69^*
Ca(umol/L)	DFDT	1.64±1.52	1.24±1.24	1.09±0.98	1.05±0.89	1.01±0.87	0.83±0.79	0.94±0.92
	GFGT	1.64±1.52	1.38±1.37	1.63±1.58	1.10±1.04	0.72±0.71	0.84±0.92	0.78±0.59
K(mmol/L)	DFDT	18.81±9.26	15.56±8.27	14.33±7.59	13.60±6.29	14.78±7.04	11.89±7.00	13.63±7.90
	GFGT	18.81±9.26	16.46±8.23	19.26±13.02	15.49±7.98	11.22±7.06	13.38±8.84	13.72±6.79

Values are represented as means±standard deviation. ^*p<0.05.

DFDT: Freeze directly and thaw directly; GFGT: Freeze by temperature gradient and thaw by temperature gradient.

Baseline represents fresh urine.

Discussion

The data demonstrate that total protein, calcium, and potassium concentrations in urine are stable when frozen and thawed up to five times with storage at either −20°C or −80°C. Our results show that albumin can only sustain three freeze/thaw cycles at −20°C using the DFDT protocol which is different than that found by Bao et al.⁷; however, our results also show that albumin was stable using the DFGT protocol for five freeze/thaw cycles at −20°C. We recommend that urine be stored at −80°C as albumin and the organic ions evaluated here are relatively stable through five and six freeze/thaw cycles, respectively, using the simple DFDT protocol. Our results also show that in most cases gradient freeze/thaw cycles are not necessary for urine sample storage.

Footnotes

Author Disclosure Statement

No competing financial interests exist. This work was supported by Shanghai Science and Technology funds (12DZ2295004).

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