Collection, Processing, and Storage Consideration for Urinary Biomarker Research

Summary

This protocol aims to provide considerations for urine sample collection, processing, and storage for urine tract infection biomarker studies.

Abstract

There are several urinary proteins that show promise as novel markers of urinary tract infections. The identification of a novel biomarker that has greater predictive accuracy compared to current diagnostic methods has the potential to greatly improve the ability to manage patients with urinary tract infections. However, sample collection, processing, and storage can all potentially impact the results of biomarker research. Understanding the effects of each of these stages on biomarker studies is necessary to inform future, high-quality research in this area, as well as to critically review other studies in this area. Here, the study reviews the literature regarding the effects of each stage of urine sample processing and reports the effects of various conditions on urinary proteins. The protocol will focus on collection techniques, time and temperature of storage, processing techniques, use of reagents, and long-term freezing on biomarker stability. It will focus on proteins but will briefly discuss other materials that may be utilized in biomarker research. In doing so, this protocol will provide a guide to future researchers to assist in the design of urinary biomarkers studies.

Introduction

Urinary tract infections (UTI) are one of the most common bacterial infections in both children and adults¹. While the diagnosis of UTI in some populations can be uncomplicated, it can be more complex in others, such as those with neuropathic bladders². The ability to accurately diagnose UTIs will help improve antibiotic stewardship efforts by decreasing the use of unnecessary antibiotics and potentially aid in the earlier diagnosis of UTI, thus decreasing the risk of morbidity. Given the prevalence of UTIs, there is significant interest in improving the management of this common infection.

There is an increasing number of novel biomarkers within the literature that show promise in their ability to diagnose UTI^3,4,5,6,7. However, there are several factors associated with the processing of urine samples that have the potential to alter results. These factors range from collection methods, temperature and duration of short and long-term storage, processing techniques, reagent use, and freeze-thaw cycles⁸. Understanding how changes in each of these can affect biomarker readings is necessary to both critically interpret research within the literature as well as design high-quality studies focused on urine biomarkers.

Here, a narrative review of the literature is provided on the effects of each factor, including collection techniques, short and long-term storage temperature and duration, reagent use, and the effect of freeze-thaw cycles, on proteins that may have utility as urine biomarkers and provide recommendations for optimal processing based on this review of the literature. This protocol will focus on protein biomarkers measured using western blots or ELISAs.

Protocol

This protocol follows the guidelines of the institution's human research ethics committee. Ensure that the approval is obtained from the institutional review board (IRB) prior to the collection and utilization of biological specimens for research.

1. Collection

1. Obtain urine sample in a sterile specimen cup. Decide the type of urine sample, as well as specific inclusion and exclusion criteria, based on the specific study design. For UTI studies, use either the clean-catch method or catheterization to avoid perineal contamination.
2. To obtain a clean-catch urine sample, instruct the participants to wipe down the periurethral area with a towelette, void a small amount into the toilet, and then urinate into the specimen cup.
3. Instruct women to use their fingers to spread the labia and men to retract their foreskin (if applicable) prior to urination to avoid contamination.
4. Record the time of collection.
5. Collect the relevant clinical data from each participant, as required by individual study design and research question.
6. Consider performing a urinalysis or urine dipstick on each sample prior to processing and storage if this data is not reliably available from the electronic health record.

2. Sample processing and storage

1. Process the samples immediately. If this is not possible, store the sample at 4 °C for up to 24 h.
2. If samples cannot be stored at 4 °C or need to be stored at 4 °C longer than 24 h, add 0.2 M boric acid or 10 mM NaN₃ to the samples. Check to ensure such reagents are compatible with planned downstream applications.
3. Record the duration of time samples spent at 4 °C.
4. Centrifuge the samples at 1000-1500 x g for 10-20 min. Centrifugation does not need to be at 4 °C.
5. Collect the supernatant and aliquot it into separate microcentrifuge tubes.
6. Label the tubes with multiple, clear, identifiers (such as the date and sample identification (ID)). Consider using computer-generated barcodes specifically designed for storage of biological samples at -80 °C. If unavailable, ensure that the pen used to label samples is water-resistant.
7. Label each freezer box such that each location has a specific code. For this, number each column with a different letter and each row with a number. This will allow for the creation of maps or other guides for easy sample location.
8. Freeze the samples immediately at -80 °C. Record the time of freezing.
9. Thaw the samples in a 37 °C water bath on the day of measurement to minimize unnecessary storage at either room temperature or 4 °C.
10. Record the times and number of additional freeze-thaw cycles for each aliquot.

3. Analysis

1. When using commercially available ELISAs, follow the manufacturer's instructions.
2. Run the samples in duplicate.
3. Identify the expected concentration of the protein of interest to ensure that the protein levels in the samples fall within the range of the kit. If the expected level of protein exceeds the upper standard, dilute the samples.
4. After data is obtained from the plate reader (ELISA) or western blot, determine the concentration of each biomarker in the sample manually (not recommended) or using any software.
5. Analyze the results. Data analysis depends on the individual study design.
6. Consider adjusting biomarker values to account for the urine concentration.
   NOTE: Traditionally, biomarker researchers have used urine creatinine as a method of normalization, especially in participants with normal renal function, to account for urine concentration. However, others report that normalization does not make a difference in the results⁴. To overcome this hurdle, some researchers report both normalized and non-normalized results.
7. Recommend reporting ranges of times from collection to freezing, as well as the duration of time at 4 °C prior to processing in published manuscripts to allow for interpretation of results in the context of sample processing (Figure 1)

4. Effect of various storage conditions on neutrophil gelatinase-associated lipocalin (NGAL).

1. Spike fresh urine with 2 ng/mL of recombinant NGAL.
2. Aliquot the urine and subject it to different processing and storage conditions.
   1. Centrifuge the urine at 1000-1500 x g for 10-20 min. Centrifugation does not need to be at 4 °C. Store at different conditions (20 °C, 4 °C, -20 °C) for 24 h, 48 h, or 72 h.
   2. Store aliquot of the sample at -80 °C for comparison.
3. After maintaining the samples in the different conditions as mentioned in step 4.2.1, measure the levels of NGAL in the samples using a commercially available ELISA kit which includes the controls as per the manufacturer's instructions.

Results

Centrifugation had a small impact on NGAL levels. Centrifuged samples stored at -80 °C had lower levels of NGAL than non-centrifuged samples (2.17 ng/mL ± 0.32 ng/mL, 2.77 ng/mL ± 0.21 ng/mL). Freeze cycles also had an impact on NGAL levels after the third freeze-thaw cycle. (Figure 2). Of the conditions studied (centrifugation, freeze-thaw cycles, and storage temperature), storage temperature had the greatest impact on NGAL levels. Storage at -80 °C immediately after sam...

Discussion

The importance of producing consistent and reproducible results is not limited to the success of individual studies but will also enable a better comparison of results within the literature⁹. Variation between studies in key procedural steps can introduce irreversible bias that may affect biomarker signals and their interpretation, which may be responsible for discrepancies among several studies¹⁰. This demonstrates the need to establish a more standardized approach to proc...

Materials

Name	Company	Catalog Number	Comments
Boric acid	Sigma-Aldrich	B6768	To be considered for samples that cannot be rapidly processed and frozen
Freezer boxes	Fisher Scientific	03-395-464
Microcentrifuge tubes	Thomas scientific	1149X93
NGAL ELISA Kit	R&D Systems	DLCN20	Used to create representative results
Pipette and tips	Dependent on pipette size and volume of fluid.
Sodium azide	Sigma-Aldrich	S2002	To be considered for samples that cannot be rapidly processed and frozen
Urine collection cups	Thermo Scientific	3122B03ORG	Sterile cups not required unless needed for other studies