Assessment of Plasma Oxalate Concentration in Patients With CKD

Abstract

Introduction: Alterations in oxalate homeostasis are associated with kidney stone disease and progression of chronic kidney disease (CKD). However, accurate measurement of plasma oxalate (P_Ox) concentrations in large patient cohorts is challenging as prompt acidification of samples has been deemed necessary. In the present study, we investigated the effects of variations in sample handling on P_Ox results and examined an alternative strategy to the established preanalytical procedures.

Methods: The effect of storage time at room temperature (RT) and maintenance of samples at -80°C was tested. P_Ox was measured in 1826 patients enrolled in the German Chronic Kidney Disease (GCKD) study, an ongoing multicenter, prospective, observational cohort study.

Results: We demonstrate that P_Ox concentrations increased rapidly when samples were maintained at RT. This was most relevant for P_Ox <10 μM, as concentrations more than doubled within a few hours. Immediate freezing on dry ice and storage at -80°C provided stable results and allowed postponement of acidification for >1 year. In the patients of the lowest estimated glomerular filtration rate (eGFR) quartile, median P_Ox was 2.7 μM (interquartile range [IQR] <2.0-4.2) with a median eGFR of 25.1 ml/min per 1.73 m² (IQR 20.3-28.1).

Conclusion: We conclude that immediate freezing and maintenance of plasma samples at -80°C facilitates the sample collection process and allows accurate P_Ox assessment in large cohorts. The present study may serve as a reference for sample handling to assess P_Ox in clinical trials and to determine its role in CKD progression.

Keywords: POx concentration; POx measurement; chronic kidney disease; clinical trials; preanalytical conditions.

Graphical abstract

Figure 1

Measurement of oxalate concentrations in plasma versus serum. Oxalate concentrations were measured in plasma (P_Ox) and serum (S_Ox). For each patient, an EDTA tube and a serum tube were drawn at the same time, immediately put on ice and processed. (a) S_Ox concentrations tend to be slightly higher than P_Ox concentrations (n = 10, P = 0.10, mean coefficient of variation 0.10). The red line indicates the mean values of all P_Ox/S_Ox concentrations. (b) S_Ox/P_Ox ratio depending on the P_Ox concentration.

Figure 2

Plasma (P_Ox) and serum (S_Ox) oxalate concentrations following storage at room temperature (RT). Oxalate concentrations were measured in plasma (a–c) and serum (d–f) samples with a concentration range of P_Ox/S_Ox < 10 μM (a+d), P_Ox/S_Ox 10–25 μM (b+e), and P_Ox/S_Ox >25 μM (c+f). For each patient, 4 to 5 EDTA or serum tubes were drawn. Each tube was processed at a different timepoint following storage at RT as indicated. Each black line represents P_Ox/S_Ox results of one patient. The red line indicates the mean values of all P_Ox/S_Ox concentrations. (a–c) The P value compares P_Ox concentrations at 0 h versus 6 h (a: n = 5, P = 0.006; b: n = 6, P = 0.07, mean coefficient of variation [CV] 0.08; c: n = 6, P = 0.051, mean CV 0.04). (d–f) The P value compares S_Ox concentrations at 0 h versus 6 h (d: n = 3, P = 0.02; e: n = 6, P = 0.12, mean CV 0.06; f: n = 6, P = 0.35, mean CV 0.03).

Figure 3

Plasma oxalate (P_Ox) measurement following freezing on dry ice and storage at −80 °C. (a) P_Ox concentrations were measured immediately after blood draw. Aliquots of unprocessed plasma were stored on dry ice and at RT. After 6 hours, P_Ox concentrations were measured and compared with P_Ox concentrations obtained at 0 h. The blue lines indicate P_Ox concentrations after storage on dry ice (n = 5, P = 0.06), the black lines indicate P_Ox results after storage at RT (n = 5, P = 0.002). The red lines indicate the mean values of P_Ox concentrations maintained on dry ice (lower line) versus at room temperature (RT) (upper line). (b,c) P_Ox concentrations were measured immediately after blood draw and following storage at −80 °C and compared via P value. Each blue line represents P_Ox results of 1 patient after storage at −80 °C. The red line indicates the mean value of all P_Ox concentrations. (b) Unprocessed plasma was compared (n = 12, P = 0.04, mean coefficient of variation [CV] 0.10) with (c) eluate obtained following protein separation and acidification (n = 12, P = 0.13, mean CV 0.08).

Figure 4

Schematic of standard and simplified test protocol for measurement of plasma oxalate (P_Ox) concentration. The preanalytical steps for processing of blood samples to determine P_Ox concentration are shown. (a) According to the standard test protocol, blood samples need to be centrifuged and the supernatant acidified within a total of 2 hours. In contrast, we demonstrate that (b) the test protocol can be simplified by placement of plasma on dry ice followed by storage at −80 °C. This prevents addition of high-molarity HCl in outpatient settings, and allows collection of large amounts of samples for efficient processing with low intra-and interassay variability.

Figure 5

Plasma oxalate (P_Ox) concentrations and corresponding estimated glomerular filtration rate (eGFR) levels in 1826 patients with chronic kidney disease (CKD) (German Chronic Kidney Disease [GCKD] study population). P_Ox concentrations were measured in 1826 patients of the GCKD study and plotted against the corresponding calculated eGFR. Values below the validated lower limit of detection of 2 μM (red dashed line) were set to 1.9 μM. The y-axis is log-scaled.