Appendix G

Stanbio Glucose LiquiColor® Procedure No. 1070

Summary and Principle

Measurement of blood glucose levels was among the first chemical procedures employed in clinical laboratory medicine.1 Glucose oxidase methodology was introduced by Keilin and Hartree in 1948. Keston later reported use of the combined glucose oxidase- peroxidase reagent, followed by the Teller addition of a chromogenic reagent to Keston's procedure. The Stanbio single reagent glucose method is based on a technique described by Trinder et al.

Glucose is oxidized in the presence of glucose oxidase (GO). The hydrogen peroxide formed reacts, under the influence of peroxidase (PO), with phenol and 4-aminoantipyrine to form a redviolet quinone complex. The intensity of the color is proportional to glucose concentration.

Glucose + O2 + H2O                        ------->  Gluconic Acid + H2O2
H2O2 + 4-aminoantipyrine + Phenol  ------->Quinone Complex

Reagents

• Glucose LiquiColor® Reagent, Cat. No. 1071 Contains:
  • 4-aminoantipyrine   0.2 mmol/L
  • Glucose Oxidase  15.0 U/mL
  • Peroxidase             1.2 U/mL
  • Phenol                   4.0  mmol/L
  • Non reactive ingredients and preservatives
• Glucose Standard, (100 mg/dL), Cat. No. 1072 
  • Glucose in aqueous benzoic acid.

Precautions: For In Vitro Diagnostic Use.

• Reagent Preparation:
  • Reagent and standard are ready-to-use.
• Reagent Storage and Stability:
  • Reagent is stable, when stored at 2-8°C until expiration date on the label. 
  • Once opened, contamination must be avoided.
• Standard is stable until expiration date on label when stored at 15-30°C.
• Reagent and standard should beat room temperature before use.
  NOTE: To prevent contamination of Glucose reagent, pour into a separate vessel a volume slightly in excess of that required.  DO NOT RETURN UNUSED PORTION.

Materials Required But Not Provided

• Spectrophotometer capable of reading at 500 nm (492-530 nm)
• Accurate pipetting devices
  Heating block or water bath, 37°C
• Cuvets
• Vortex Mixer
• Interval Timer

Specimen Collection and Preparation

• Serum:
• Remove from clot within 30 minutes of collection in order to prevent glycolysis.
• Plasma:
  • An anticoagulant containing fluoride is recommended, but any of the common anticoagulants may be used if plasma is separated from cells promptly after centrifugation.
• CSF:
  • No special preparation is required.
• Sample Stability:
  • Glucose in serum/plasma processed in the manner described is stable for 40 hours at 2-8°C.
  • CSF samples should be analyzed immediately because of possible bacterial contamination.
• Interfering Substances:
  • False low glucose values can result from excessive levels of ascorbic acid.
    
    

Manual Procedure

• Pipet into cuvets the following volumes (mL) and mix well:

	Reagent blank	Standard (S)	Sample (U)
Reagent	1.0	1.0	1.0
Standard	-	0.01	-
Sample	-	-	0.01

• NOTE: Volumes may be increased if the instrument requires a volume greater than 1.0 mL.
• Incubate all cuvets at 37°C for 5 minutes.
• Read S and U vs RB at 500 nm within 30 minutes.

Quality Control: Two levels of control material with known
glucose levels determined by this method or a hexokinase procedure
should be analyzed each day of testing. Stanbio recommends,
Ser-T-Fy® I, Normal Control and Ser-T-Fy® II, Abnormal
Control be assayed with each patient run.

Results

• Values are derived by the following equations:
  Glucose (mg/dL) =  Au/As x 100,
  
• where Au and As are the absorbance values of UNKNOWN and STANDARD, respectively
• and 100 the concentration of the STANDARD (mg/dL).
• Example: Following readings were obtained using 1 cm cuvets:
  • Au = 0.130, As = 0.178  Glucose (mg/dL) = x 100 = 73
  • Glucose (mmol/L) = Glucose (mg/dL) x 0.0556

NOTE: Samples having glucose values greater than 500 mg/dL are diluted 1:2 (1 + 1) with distilled water, the assay repeated and results multiplied by the dilution factor 2.

Expected Values

• Normal Range: Serum/Plasma:70-105 mg/dL(3.9-5.8mmol/L)
• CSF: 40-75 mg/dL (2.2-3.9 mmol/L)
• It is recommended that each laboratory establish its own range of
  expected values, since differences exist between instruments, laboratories, and local populations.
  

Performance Characteristics

Percision

Using a serum containing glucose in the normal range and one with elevated values, a series of 5 assays were performed on each of 5 days. Coefficients of variation (CV) were within run of 1.6 and 1.2%, and between runs 3.0 and 2.0%, respectively.

Correlation:

Determination of glucose by the procedure described (y) and by a hexokinase/UV method (x) on 40 sera (range: 56-582 mg/dL) showed a correlation coefficient (r) of 0.995 and a regression equation of y = 0.98x -1.99.

Linearity:

When performed as directed the method is linear from 0 to 500 mg/dL.

References

• 1.Folin O., Wu H.: J Biol Chem 41:367, 1920
• 2.Keilin D., Hartree E.F.: Biochem J 42:230, 1948
• Keston AS: Abstr 129th Meeting, Am Chem Soc, 1956, p 31c.
• Teller J.D.: Abstr 130th Meeting, Am Chem Soc, 1956, p 69c.
• Trinder, P., "Determination of Blood Glucose Using
• Aminophenazone." J. Clin. Path., 22:246 (1959).
• Howanitz P.J., Howanitz J.H.: IN Clinical Diagnosis and Management by Laboratory Methods, 17th ed., J.B. Henry, Ed., W.B. Saunders, Philadelphia, 1984, p 168.
• Cooper G.R., McDaniel V: Manual of Methods for the Determination of Glucose, CDC, USPHS, Atlanta.
• Caraway W.T.: IN Fundamentals of Clinical Chemistry, 2nd ed., N.W. Tietz, Ed. Saunders, Philadelphia, 1976, p 242.
• Stanbio Laboratory data

Walter I. Hatch
wihatch@smcm.edu

August 12, 2012