SUMMARY AND EXPLANATION

Creatinine is the end product of creatine metabolism which occurs mostly in skeletal muscle. Since creatinine undergoes glomerular filtration but is not reabsorbed by the renal tubes, its level in serum provides a useful index of renal function (1).

Creatinine determinations usually involve the Jaffe reaction or one of its many modifications (2). The DIRECT CREATININE PROCEDURE is  based on the Jaffe reaction.

PRINCIPLE

In the Jaffe reaction, creatinine reacts with picric acid under alkaline conditions to form a red-colored product that can be measured kinetically at 510 nm. Change in red color intensity is directly proportional to creatine concentration.

REAGENTS: FOR IN-VITRO DIAGNOSTIC USE

Reagent Set Cat. No. 2810 provides:

CREATININE PICRIC ACID REAGENT - (Cat. No. 2811)

REACTIVE INGREDIENTS:

33mM picric acid.

PRECAUTIONS:

Cause irritation. Avoid contact with eyes, skin and clothing, In case of contact, wash with large amount of water.

STORAGE AND STABILITY:

Store at 15-30ºC. Stable until expiration date if sealed tightly.

DETERIORATION:

The reagent should be a clear, yellow solution. An orange-red color or cloudiness would indicate deterioration.

CREATINE BASE REAGENT - (Cat. No. 2812)

REACTIVE INGREDIENTS:

0.4 m sodium hydroxide.

PRECAUTIONS:

Causes irritation. Avoid contact with eyes, skin and clothing. In case of contact, wash with large amount of water.

STORAGE AND STABILITY:

Store at 15-30ºC. Stable until expiration date if sealed tightly.

DETERIORATION:

The reagent should be a clear, colorless solution.

CREATININE CALIBRATOR - (Cat. No. 2813)

REACTIVE INGREDIENTS:

Creatinine hydrochloride equivalent to 5.0 mg/dL serum creatinine.

PRECAUTIONS:

Causes irritation. Avoid contact with eyes, skin and clothing. In case of contact, wash with large amount of water.

STORAGE AND STABILITY:

Store at 15-30ºC. Stable until expiration date if sealed tightly.

DETERIORATION:

The calibrator is a clear, colorless solution. Turbidity would indicate deterioration.

INSTRUMENTS

Use a spectrophotometer or colorimetric calibrated at 510 nm.

SPECIMEN COLLECTION

PRECAUTIONS:

1. Use of unhemolyzed serum is recommended

2. 24 hour urine collections should be preserved with 15 g boric acid

3. Avoid microbial contamination

SAMPLE STORAGE:

Serum creatinine appears stable for one week at 2-8ºC or three months frozen.

ADDITIVES:

No special additives or preservatives are needed for serum. Urine collections should be preserved with 15 g boric acid.

INTERFERING SUBSTANCES:

Young et al. (3) have reviewed drug effects on serum and urine creatinine. Grossly lipemic or hemolyzed samples should not be used.

PROCEDURE

MATERIALS PROVIDED:

CREATININE PICRIC ACID REAGENT (Cat, No. 2511), CREATININE BASE REAGENT (Cat. No. 2612), and CREATININE CALIBRATOR (Cat. No. 2613).

MATERIALS REQUIRED BUT NOT PROVIDED:

1. 0.05 mL micropipettor

2. 1.0 mL pipettor or dispenser

3. 37ºC Incubator

REACTION CONDITIONS:

Wavelength 510 nm

Incubation Time 1 min

Incubation Temperature 37º C

            Sample Volume                         0.05 mL

Picric Acid Reagent Volume   0.5 mL

Base Reagent Volume 0.05 mL

Total Volume 1.05 mL

Low Normal 0.5  mg/dL

High Normal 1.6  mg/dL

Calibrator value 5.0  mg/dL

PERFORMANCE OF TEST

WORKING REAGENT PREPARATION:

Prepare the working reagent by adding one part Picric Acid Reagent to one part Base Reagent and mixing well. This working reagent is stable for eight hours at room temperature.

NOTE: Dilute urine samples X 10 with D-H20 prior to assay.

1.   Label a series of tubes CALIBRATOR, CONTROL, SAMPLE 1, etc.

2.   Dispense 1.0 mL working reagent into each tube. (Alternately, add 0.5 mL Picric Acid Reagent and 0.5 mL Base Reagent to each tube).

3. Prewarm tubes with Reagent to 37ºC.

4. Zero instrument at 510 nm with D-H20.

5. For each sample (Calibrator, Control, Unknown), perform the following:

a. Add 0.05 mL sample into appropriate tube and mix well.

b. Incubate at 37ºC for 20 seconds and read absorbance (A1).

c. Incubate at 37ºC for an additional 80 seconds and read absorbance (A2).

CALIBRATION:

It is not necessary to perform a calibration curve with this procedure since the reaction is linear in the range of 0-20.0 mg/dL. However, a Calibrator must be determined with each set of Unknowns assayed. Use CREATININE (KINETIC) CALIBRATOR (Cat. No. 2603) which is provided with the reagent set or other suitable calibration material for this purpose. Specimens exceeding 20.0 mg/dL should be diluted with 0.9% sodium chloride and reassayed. Multiply the test result by the dilution factor to obtain final answer.

QUALITY CONTROL:

the reliability of test results should be monitored routinely using suitable quality control materials (normal and abnormal) analyzed in the same manner used for the Unknowns. Eagle Diagnostics offers CHEM-TROL NORMAL (Cat. No. 8100) and CHEM-TROL ELEVATED (Cat. No. 8200) for this purpose. Failure to achieve assayed values of freshly prepared control sera must be thoroughly investigated before patient results are reported.

CALCULATION OF RESULTS

The following equations are used to determine unknown values:

SERUM:

Unk Conc   =

(A2 - A1) Unk

-------------   x  Cal Conc (mg/dL)

(A2 - A1) Cal

EXAMPLE:

A 5.0 mg/dL calibrator had A2 - A1  = 0.120 while the unknown had A2 - A1  =  0.417. The creatinine concentration of the unknown is:

0.147

------  x 5.0 mg/dL  =  6.1 mg/dL

0.120

URINE:

Urine Creatinine (g/24 hours) =

(A2 - A1) Unk

-------------  x Cal Conc (mg/dL)

(A2 - A1) Cal

x  0.01   x   10  x 24 hr urine volume (L)

The factor 0.01 converts mg/dL while the factor 10 adjusts for urine dilution prior to assay.

EXAMPLE:

The 10x diluted urine had A2 - A1  =  0.149 and the 24 hour urine collection volume  =  1800 mL. The excreted creatinine is:

0.149

-------  x  5.0 mg/dL   x  0.01  x  10  x  1.8 L/24 hr

0.120

=  1.12 g/24 hour

CREATININE CLEARANCE:

Creat. Clear (mL/min) =
 
 

Urine Conc (mg/dL) X Urrine Vol (mL/min)

------------------------------------------

Serum Conc (mg/dL)
 
 

EXAMPLE:

From this data above,

Urine Conc =

0.149

------  x 5.0 mg/dL X 10 = 6.21 mg/dL

0.120

Urine Vol = 1800 mL/1440 min = 1.25 mL/min

Serum Conc. = 6.1 mg/dL

6.21 mg/dL X 1.25 mL/min

--------------------------   = 13 mL/min

6.1 mg/dL

EXPECTED VALUES

SERUM: 0.5-1.6 mg/dL

URINE:   0.6-1.6 g.24 hr.

CREATININE CLEARANCE

MALES: 105 + 20 mL/min

FEMALES:  95 + 20 mL/min

PERFORMANCE CHARACTERISTICS

PRECISION:

Normal and abnormal control sera were assayed 20 times each to establish within run precision and for 10 working days to establish run to run precision.

MEAN / STD.DEV. / %CV

WITHIN RUN

Normal 1.3 / 0.04 / 3.1

Abnormal 5.2 / 0.03 / 0.6
 
 

RUN TO RUN

Normal 1.4 / 0.06 / 4.3

Abnormal 5.1 / 0.04 / 0.8

SPECIFICITY:

A comparison of the CREATININE (KINETIC) PROCEDURE with another widely used commercial method showed a 98% correlation with 42 serum samples in the normal and abnormal range.

SENSITIVITY:

The CREATININE (KINETIC) PROCEDURE has sensitivity of 0.042 mg/dL per 0.001 absorbance units.

REFERENCES

1. Tietz, N.W., Fundamentals of Clinical Chemistry, 2nd ed.. W.B.Saunders Co., Philadelphia, 1982, p. 994.

2. Slot, C., Scand. J. Clin. Lab. Invest. 17,381 (1965).

3. Young, D.S., Pestaner, L.C., and Gibberman, V., Clin. Chem. 21, 286D (1975).