2.14: Serum Creatinine (Jaffe Method)
- Page ID
- 38642
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\(\newcommand{\avec}{\mathbf a}\) \(\newcommand{\bvec}{\mathbf b}\) \(\newcommand{\cvec}{\mathbf c}\) \(\newcommand{\dvec}{\mathbf d}\) \(\newcommand{\dtil}{\widetilde{\mathbf d}}\) \(\newcommand{\evec}{\mathbf e}\) \(\newcommand{\fvec}{\mathbf f}\) \(\newcommand{\nvec}{\mathbf n}\) \(\newcommand{\pvec}{\mathbf p}\) \(\newcommand{\qvec}{\mathbf q}\) \(\newcommand{\svec}{\mathbf s}\) \(\newcommand{\tvec}{\mathbf t}\) \(\newcommand{\uvec}{\mathbf u}\) \(\newcommand{\vvec}{\mathbf v}\) \(\newcommand{\wvec}{\mathbf w}\) \(\newcommand{\xvec}{\mathbf x}\) \(\newcommand{\yvec}{\mathbf y}\) \(\newcommand{\zvec}{\mathbf z}\) \(\newcommand{\rvec}{\mathbf r}\) \(\newcommand{\mvec}{\mathbf m}\) \(\newcommand{\zerovec}{\mathbf 0}\) \(\newcommand{\onevec}{\mathbf 1}\) \(\newcommand{\real}{\mathbb R}\) \(\newcommand{\twovec}[2]{\left[\begin{array}{r}#1 \\ #2 \end{array}\right]}\) \(\newcommand{\ctwovec}[2]{\left[\begin{array}{c}#1 \\ #2 \end{array}\right]}\) \(\newcommand{\threevec}[3]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \end{array}\right]}\) \(\newcommand{\cthreevec}[3]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \end{array}\right]}\) \(\newcommand{\fourvec}[4]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \\ #4 \end{array}\right]}\) \(\newcommand{\cfourvec}[4]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \\ #4 \end{array}\right]}\) \(\newcommand{\fivevec}[5]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \\ #4 \\ #5 \\ \end{array}\right]}\) \(\newcommand{\cfivevec}[5]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \\ #4 \\ #5 \\ \end{array}\right]}\) \(\newcommand{\mattwo}[4]{\left[\begin{array}{rr}#1 \amp #2 \\ #3 \amp #4 \\ \end{array}\right]}\) \(\newcommand{\laspan}[1]{\text{Span}\{#1\}}\) \(\newcommand{\bcal}{\cal B}\) \(\newcommand{\ccal}{\cal C}\) \(\newcommand{\scal}{\cal S}\) \(\newcommand{\wcal}{\cal W}\) \(\newcommand{\ecal}{\cal E}\) \(\newcommand{\coords}[2]{\left\{#1\right\}_{#2}}\) \(\newcommand{\gray}[1]{\color{gray}{#1}}\) \(\newcommand{\lgray}[1]{\color{lightgray}{#1}}\) \(\newcommand{\rank}{\operatorname{rank}}\) \(\newcommand{\row}{\text{Row}}\) \(\newcommand{\col}{\text{Col}}\) \(\renewcommand{\row}{\text{Row}}\) \(\newcommand{\nul}{\text{Nul}}\) \(\newcommand{\var}{\text{Var}}\) \(\newcommand{\corr}{\text{corr}}\) \(\newcommand{\len}[1]{\left|#1\right|}\) \(\newcommand{\bbar}{\overline{\bvec}}\) \(\newcommand{\bhat}{\widehat{\bvec}}\) \(\newcommand{\bperp}{\bvec^\perp}\) \(\newcommand{\xhat}{\widehat{\xvec}}\) \(\newcommand{\vhat}{\widehat{\vvec}}\) \(\newcommand{\uhat}{\widehat{\uvec}}\) \(\newcommand{\what}{\widehat{\wvec}}\) \(\newcommand{\Sighat}{\widehat{\Sigma}}\) \(\newcommand{\lt}{<}\) \(\newcommand{\gt}{>}\) \(\newcommand{\amp}{&}\) \(\definecolor{fillinmathshade}{gray}{0.9}\)RELATED READING: Chapter 26. See Methods in CD-ROM for Creatinine.
OBJECTIVES
Upon completion of this exercise, appropriate discussion, and related readings, the student will be able to:
- Explain the principle of protein-free filtrate preparation.
- Prepare a protein free filtrate of serum.
- Explain why a filtrate preparation is required in procedures of this type.
- Determine the creatinine value of 2 unknown samples with 90% accuracy.
- List substances that cause interferance with the alkaline picrate method of creatinine analysis.
PRINCIPLE
Creatinine reacts with picric acid in an alkaline solution to form a reddish colored complex. The reaction is commonly known as the Jaffe reaction and the red colored product as the Janovski complex.
MATERIALS
- 16 x 100 mm Test Tubes
- Creatinine Standard
- Picric Acid Reagent
- Controls
- Sodium tungstate
- Patient Samples
- Sulfuric Acid
- Spectrophotometer
- Distilled Water
- Sodium Hydroxide
- Table Top Centrifuge
PROCEDURE
- Label 16 x 100 mm test tubes for each control and patient sample to be tested.
- To each control and patient sample tube, add 1 mL sodium tungstate reagent, 1mL sulfuric acid reagent, and imL of distilled water. Mix well.
- Add 1 mL of each control or patient serum to the appropriate tube.
- Mix well and centrifuge for 5 minutes at 1500 RPM.
- Label clean 16 x 100 mm test tubes for a blank, standard, and each control or sample for which a “filtrate” was prepared.
- Add reagents/samples to each tube according to the following chart. It is acceptable to add picric acid reagent and NaOH sequentially to all tubes after all other materials have been added. Each test tube should contain the same final volume.
| Distilled Water | Working Standard | Filtrate | Picric Acid | NaOH | |
|---|---|---|---|---|---|
| Reagent Blank | 4 | 0 | 0 | 1 | 1 |
| Standard | 3.5 | 0.5 | 0 | 1 | 1 |
| Control (s) | 2 | 0 | 2 | 1 | 1 |
| Patient Sample | 2 | 0 | 2 | 1 | 1 |
- Mix well and allow to stand for 15 minutes at room temperature.
- Transfer the contents of test tube to an appropriate cuvette and read the absorbance at 510 nm against the blank solution.
- Record the results on the data sheet.
- Determine the creatinine values for the control and patient samples by proportional calculation (see Exercise #1) using the concentration of the standard and it’s absorbance.
OPTIONAL EXERCISE
Add a 0.10 mL of a solution of acetone (80 g/L) to 2.0 mL of a control or patient sample while mixing. Test solution for ketones by dipsticks. Assay unspiked and spiked samples for creatinine. Record results on data sheet.
| DATA SHEET, EXERCISE #14 |
NAME: ___________ DATE: ___________ |
RESULTS
| Absorbance | Creatintine Value (mg/L) | Target Value | |
|---|---|---|---|
| Creatinine Standard | |||
| Normal Control | |||
| Abnormal Control | |||
| Pt # | |||
| Pt # |
Optional Exercise
| Unspiked sample | ||
| Acetone-spiked sample |
Discussion Questions
- What substances will cause interference in the alkaline picrate method?
- What substances will interfere when using a protein free filtrate?
- What problems will arise if the procedure is carried out at a temperature above room temperature?
- Do you suspect that any of the samples you tested may have shown positive interference? How could this be dealt with?
- In the optional exercise, was there any interference by the added acetone? Might there have been an interference if the reaction had been performed in the kinetic or endpoint mode without first preparing a protein-free filtrate?
- Was the method in this experiment an end—point or kinetic procedure?