CREATININE (DIRECT) PROCEDURE

Intended for the Quantitative Determination of Creatinine in Serum and Urine

SUMMARY AND EXPLANATION

Creatinine is the end product of creatnine metabolism which occurs mostly in skeletal muscle. Since creatinine undergoes glomerular filtration but is not reabsorbed by the renal tubules, 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 EAGLE DIRECT CREATININE PROCEDURE is based on the Heinegard and Tiderstrom (3) modification of the Jaffe reaction in which a surfactant and borate are used to reduce interference from protein and glucose.

PRINCIPLE

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

REAGENTS: FOR IN-VITRO DIAGNOSTIC USE

Reagent Set Cat. No. 2600 provides:

PICRIC ACID REAGENT - (Cat. No. 2601)

REACTIVE INGREDIENTS:

3.3 mM picric acid

PRECAUTIONS:

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

BASE REAGENT - (Cat. No. 2602)

REACTIVE INGREDIENTS:

0.17 M sodium hydroxide and 26 mM sodium tetraborate. Surfactant added.

PRECAUTIONS:

Causes irritation. Avoid contact with eyes, skin and clothing. In case of contact, wash with large amounts 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. NOTE: Upon chilling, a white precipitate may form. Warm at 37ºC to effect solution and mix gently until completely redissolved.

DIRECT CREATININE CALIBRATOR - (Cat. No. 2603)

REACTIVE INGREDIENTS:

Creatinine hydrochloride equivalent to 6.0 mg/dL serum creatinine.

PRECAUTIONS:

Causes irritation. Avoid contact with eyes, skin and clothing. In case of contact, wash with large amounts 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 colorimeter calibrated at 510 nm.

SPECIMEN COLLECTION

PRECAUTIONS:

1. It is recommended that unhemolyzed serum be used.

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 - 8º or three months frozen.

ADDITIVES:

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

INTERFERING SUBSTANCES:

1. Grossly lipemic or hemolyzed samples should not be used.

2. Preservatives used in many commercial controls cause a strong reduction of the Picric Acid in this method and will show falsely elevated values even in the normal levels.

3. Young et al, (4) have reviewed drug effects on serum and urine creatinine.

PROCEDURE

MATERIALS PROVIDED:

PICRIC ACID REAGENT (Cat. No. 2601), BASE REAGENT (Cat. No. 2602) and DIRECT CREATININE CALIBRATOR (Cat. No. 2603).

MATERIALS REQUIRED BUT NOT PROVIDED:

1. 0.1 mL micropipettor

2. 1.5 mL pipettor or dispenser

3. 37ºC incubator

4. Test tubes, rack and timer

REACTION CONDITIONS:

Wavelength 510 nm

Filter Selection 490 - 530 nm

Reaction Type Endpoint

Incubation Temperature 37ºC

Incubation Time 15 minutes

Sample Volume 0.1 mL

Picric Reagent Volume 1.5 mL

Base Reagent Volume 1.5 mL

Total Volume 3.1 mL

Low Normal 0.5 mg/dL

High Normal 1.7 mg/dL

AUTOMATED PROCEDURE:

This reagent set is for manual use only. KINETIC CREATININE REAGENT SET (Cat. No. 2610) is designed for automated assays.

MANUAL PROCEDURE:

WORKING REAGENT PREPARATION:

Prepare the working reagent by adding one part PICRIC ACID REAGENT to one part BASE REAGENT and mixing well. The working reagent is stable for eight hours at room temperature.

ALTERNATE METHOD:

Dispense 1.5 mL PICRIC ACID REAGENT and 1.5 mL BASE REAGENT to each tube. Mix well. Proceed with test.

PROCEDURAL NOTE:

Dilute urine samples 1:10 with D-H2O prior to assay.

1. Dispense 3.0 mL of working reagent into tubes labeled Reagent Blank, Calibrator, Control, Sample 1, etc.

2. Add 0.1 mL sample into appropriate tube and mix well. Use D-H2O as sample for reagent blank.

3. Incubate at 37ºC for 15 minutes.

4. Zero instrument at 510 nm using the Reagent Blank.

5. Read and record absorbance values for Calibrator, Control and Unknowns.

NOTE: For a direct read-out instrument, set read-out at concentration of Calibrator and read the Unknown concentrations directly.

STABILITY OF FINAL REACTION PRODUCT:

The test samples should be read within 30 minutes after color development.

CALIBRATION:

It is not necessary to perform a calibration curve with this procedure since the reaction is linear in the range of 0-20 mg/dL. However, a Calibrator and Reagent Blank must be determined with each set of Unknowns assayed. Use DIRECT CREATININE CALIBRATOR (Cat. No. 2603) which is provided in the reagent set for this purpose.

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 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 should be thoroughly investigated before patient values are reported.

CALCULATION OF RESULTS

The following equations are used to determine Unknown Values:

SERUM:

Uknown (mg/dL)    =

Unk. Abs.

--------------    X Cal. Conc. (mg/dL)

Cal. Abs.

EXAMPLE:

An 6.0 mg/dL Calibrator had Abs. = 0.233 while the Unknown Abs. = 0.147. Thecreatinine concentration of the Unknown is:

0.147

--------  X 6.0 g/dL   =   3.8 mg/dL

0.233

URINE:

Urine Creatinine (g/24 hrs)   =

Unk. Abs.

--------------  X Cal. Conc. (mg/dL)

Cal. Abs.

X   1.67   X   0.01   X   10   X   24 hr urine volume (L)

The factor 1.67 corrects for calibrator reactivity difference in an aqueous medium (urine) versus a protein medium (serum). The factor 0.01 converts mg/dL to g/L while the factor 10 adjusts for urine dilution prior to assay.

EXAMPLE:

The 10X diluted urine had Abs. = 0.290 and the 24 hour urine collection volume = 1800 mL. The excreted creatinine is:

0.290

-------- X 6.0 g/dL  X  1.67  X  0.01  X 10 X  1.8 L/24 hr  =  2.24 g/24 hr

0.233

CREATININE CLEARANCE:

Creat. Clear.   (mL/min)   =

Urine Conc. (mg/dL)

---------------------------------   X   Urine Vol. (m/dL)

Serum Conc.   (mg/dL)

EXAMPLE:

From the data above:

Urine Conc.   =

0.290

----------  X   6.0 mg/dL   X   1.67 X 10   =   125 mg/dL

0.233

Urine Vol.      =

1800 mL

-------------  = 1.25 mL/min

1440 min

Creat. Clear.  =   

125 mg/dL   X   1.25 mL/min

-------------------------------------  = 41 mL/min

    3.8 mg/dL

EXPECTED VALUES (3)

Serum 0.5 - 1.7 mg/dL

Urine 0.6 - 1.6 g/24 hr

Creatinine Clearance (3)

Males 105 + 20 mL/min

Females 95 + 20 mL/min

PERFORMANCE CHARACTERISTICS

LINEARITY:

This method is linear to 20 mg/dL.

PRECISION:

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

WITHIN RUN

MEAN / ST. DEV. / %CV

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 this DIRECT CREATININEPROCEDURE with another widely used commercial method showed a 98% correlation with samples in the normal and abnormal range.

SENSITIVITY:

This procedure has a sensitivity of 0.025 mg/dL per 0.001 absorbance unit.

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. Heinegard, D., and Tiderstrom. G., Clin. Chem. Acta 43, 305 (1973).

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