Urinalysis JAX_URI_001

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Purpose

Urinalysis is performed to monitor renal function and will be performed as part of the adult phenotyping pipeline for KOMP2 mice. Samples will be analyzed using the Beckman AU680 autoanalyzer. Levels of urinary albumin, creatinine, magnesium and glucose are measured. The albumin to creatinine ratio (ACR) will be derived. A standard curve is run for microalbumin with each set of samples and used to derive an adjusted albumin value for use in the ACR.

Experimental Design

Minimum number of mutant animals: must maintain ≥ 7 size for male and female.

Age of animals: 13 weeks

Sexual dimorphism: Yes for some of the parameters.

Equipment

1. Clinical chemistry analyzer

2. 1.5 mL Eppendorf tubes

3. Pipettes (200-1000 ul)

Procedure

Set up the clinical chemistry analyzer and perform QC analyses of the control reagents in accordance with the equipment guidelines.

Sample collection and preparation:

a. Collect the appropriate volume of urine required (70ul), for the clinical chemistry analyser being used for assessment, in a microcentrifuge tube Time of day for collection is in the morning, starting no earlier than 07:30. If required volume is not obtained the first day, repeat collection on the next day and combine with Day 1 sample for same mouse.

b. Keep whole urine samples on ice until analysis. If samples cannot be analysed immediately, keep them at 4°C until analysis.

c. Analysis of samples is optimally done on the day of collection. When not possible the samples can be stored at 2-8°C. If samples require storage for > 48 hours, freeze at -20 °C in single aliquots. All samples are allowed to come to room temperature prior to analysis.

d. Use samples undiluted or diluted to a ratio of 1:1 with deionised water if the volume is insufficient.

Notes

Urine collection is performed as a non-fasting procedure, always in the morning.

Dilution. Dilution of urine is highly discouraged, but is allowed when the total necessary amount is not obtained. If dilution is necessary then the assays should be done in one run.

 

Metadata & examples

 

Metadata

Example

Equipment Name

ID of the machine used when more than 1 is used  having same model and manufacturer. E.g. machine 1, machine 2, machine Minnie, machine Mickey Mouse, etc.

Equipment manufacturer

Manufacturer of the equipment. E.g. Olympus Diagnostics.

Equipment model

Model of the equipment. E.g.  AU400, AU680

Anaesthesia used for urine collection

The drug used for anaesthesia during urine collection. E. g. Isofluorane. Or None.

Method of urine collection

Concise description of the method used for urine collection.

Samples kept on ice between collection and analysis

Yes/No.

Sample status

Indicate if the sample were frozen (analysis on the same day of collection not possible) or fresh (analysis on the same day of collection). E.g Fresh/Frozen.

Urine dilution

Dilution is highly discouraged but if necessary indicate the extent. E.g. No/1:1.

ID of urine collection SOP

ID of the protocol followed for urine collection. Can be a center specific protocol. E.g. ESLIM_024_001.

Date and time of urine collection

Time of day for collection is in the morning, starting no earlier than 07:30. E.g. Year, month, day, time.

Date of measurement

The day of urine analysis. Year, month, day.

Experimenter ID

An ID of any format to be used coherently both inside the same procedure and for all procedures. E.g. Harw_001, or 1/2/3.

 

Data QC

These are the kits for calibrators and QC that Jax uses:

 

Test

Au #

Test kit #

Calibrator Kit #

QC Kit #

Frequency of Assay

Creatinine

12. CRE

Beckman OSR6178

Beckman DR0091

BioRad 395X (2 levels)

Calibrator and Controls assayed for every day of a test run.

MicroAlbumin

27. MALB

Beckman OSR6167

Beckman ODR3024

BioRad 395X (2 levels)

Calibrator and Controls assayed for every day of a test run.

Magnesium

20.MG

Beckman OSR6789

Beckman DR0090

BioRad 395X (2 levels)

Calibrator and Controls assayed for every day of a test run.

Glucose

92.GLUX

Beckman OSR6121

Beckman DR0090

BioRad 395X (2 levels)

Calibrator and Controls assayed for every day of a test run.

 

 

 

 

 

 

 

 

 

 

 

To create a standard curve for calculating microalbumin:

 

Sigma Mouse Albumin (A3139-10MG) Dilution Procedure to create Standard Curve.

 

1)  Reconstitute with 1 mL Reagent Grade H2O

2)  This Stock will = 1,000 mg/dL Microalbumin

3)  Make a 2 X dilution of stock (resulting in new stock solution #2 of 500 mg/dL)

     with Beckman Diluent 1 (PN# 442825)

4)  Make a 10X dilution stock # 2 ( to new stock #3 of 50 mg/dL)

5)  Perform serial dilution 1:1 of stock #3 to obtain the following series of standards for MicroAlbumin:

  • 25 mg/dL
  • 12.5 mg/dL
  • 6.25 mg/dL
  • 3.125 mg/dL
  • 1.5625 mg/dL
  • 0 (Diluent Only)

 

6)  Run standard samples on the AU680

Using the MicroAlbumin Regression template, insert the measured values for the standards and redraw the regression line. Update the value in the divisor box to reflect the value in front of x from the regression line. The formula is set to divide each measured sample value by the divisor to calculate the known concentration (mg/dL).  Then input the creatinine concentration (mg/dL) and it will calculate and convert the ratio (mg alb/g creatinine).  

 

 

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