CCHM321 LABORATORY MIDTERMS
Lesson 5: Stanbio Glucose Oxidase
Summary and Principle
The accurate estimation of glucose is
important in the diagnosis and management of
hyperglycemia and hypoglycemia.
hyperglycemia may occur as a result of
diabetes mellitus, in patient receiving
intravenous glucose fluids, or during severe
stress. Hypoglycemia may be the result of an
insulinoma, insulin administration, inborn
error of carbohydrate metabolism or fasting.
measurement of blood glucose levels was
among the first chemical procedures employed
in clinical medicine. the glucose oxidase
methodology was introduced by Keilin and
Hartree in 1948. Keyston later reported use of
the combined glucose oxidase-peroxidase
reagent, followed by the Teller addition of a
chromogenic reagent to Keyston’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 (GODase). The hydrogen peroxide
formed reacts, under the influence of
peroxidase (POD), with phenol and 4-
aminoantipyrine to form a red-violet quinone
complex. The intensity of the color is
proportional to glucose concentration.
Reagents (ready to use)
Buffer
Phenol
4-aminoantripyrine
Glucose oxidase
Peroxidase
Non-reactive ingredients
Preservatives
Glucose Standard
Contains 5.55mmol/L (100mg/dL) glucose in
0.5mol/L benzoic acid
Glucose standard: 5.55mmol/L (100mg/dL)
Reagent Storage and Stability
Reagent at 2-8ºC (can last until expiration
date. Must be protected from light and
contamination)
Standard at 2-8 ºC (can last until expiration
date)
Glucose at 2-8 ºC or -20 ºC (can last for 48hrs
at refrigerator temperature. -20*C is for long
term storage. Make sure it is sealed to avoid
contamination and evaporation of sample)
Specimen Collection and Storage
Non-hemolyzed serum
Fluoride
Interfering Substances
Ascorbic acid
Manual Procedure
1. Zero spectrophotometer at 500nm with
distilled water.
2. For each Standard and sample tubes, add
1.0ml reagent to test tubes and warm at 37 ºC
for 5 minutes.
3. Add 10µl of each sample to its respective
tubes, mix gently and return to 37ºC
incubation.
4. After 5 minutes, read and record the
absorbance of a samples.
Results
Values are derived by comparing the
absorbance of the unknown (U) with that of a
standard (S) identically treated.
Glucose (mg/dL) = Au/As x 100
Where Au and As are the absorbances of
unknown and standard, respectively, and 100
the concentration of standard (mg/dL).
Ex: Au=0/370, As=0/280
Glucose (mg/dl) = 0.370/0.280 x 100 = 132
Expected Values
Serum or Plasma = 70-105mg/dL
CSF = 40-75md/dL
these ranges should serve only as a guideline.
It is ultimately responsibility of the laboratory
to establish its own range of expected values,
since differences exist between instruments,
laboratories, and local populations.
Data and Results
Au = 0.250
As = 0.230
Computations
Glucose (mg/dL) = Au/As x 100
Glucose (mg/dL) = 0.250/0.230 x 100
Glucose (mg/dL) = 108.69 mg/dL
Interpretations
Above Reference Range
DELA CRUZ, KJ 1
 CCHM321 LABORATORY MIDTERMS
Lesson 6: Glucose Determination by Profame
Pre-Analytical Phase
Glucose is a simple sugar containing 6 carbon
atoms. Glucose is an important source of
energy in the body and the sole source of
energy for the brain. Glucose is stored in the
body in the form of glycogen; the
concentration of glucose in the blood is
maintained at around 5mmol/L (90mg/dL) by
a variety of hormones, principally insulin and
glucagon. If the blood glucose concentration
falls below this level, neurological and other
symptoms may result (hypoglycemia).
Conversely, if the blood glucose level is raised
above its normal level to 10mmol/L (around
180mg/dL), the condition of hyperglycemia
develops. This is a symptom of Diabetes
Mellitus.
Notes
Glucose is under the category of carbohydrates
and it is one of the examples of
monosaccharides.
0.0555 mg/dL converts to mmol/L
Insulin and glucagon are the hormones produced
by pancreas.
Insulin, hypoglycemic agent
Glucagon, hyperglycemic agent
Hypoglycemia – the value of glucose is below the
normal preference range.
Hyperglycemia – one of the characteristics of
diabetes mellitus.
Method
Ortho-Toluidine, Dubowski Method
Notes
Chemical Method
o Oxidation Reduction
 Alkaline copper reduction test
 Alkaline Ferric
o Condensation
 Ortho-Toluidine method
Enzymatic Method
o Glucose Oxidase
o Hexokinase
o Glucose dehydrogenase
Principle
Glucose condenses with O-toluidine in glacia
acetic acid when heated at 100*C forming N-
glycosylamine.
Glucose + 0-toluidine--> N-glycosylamine
Notes
Ortho-Toluidine method – old approach. Good
Characteristics: Simplicity, sensitivity and
accuracy
Glucose came from the sample. 0-toluidine is
the main reagent. Glycosylamine will produce
green color compound.
Mas matingkad yung color, mas marameng
glucose na present.
Thiourea
Specimen Collection and Preparation
Serum: Remove from the 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.
Materials
Spectrophotometer
Accurate pipetting devices
Heating block or water bath (100ºC)
Cuvettes
Interval timer
Reagent Kits
For Glucose Reagent: Gluco-Toluidine
Reagent, and Acetic Acid.
For Standard: Glucose Standard, 100mg/dL
Reagent Storage and Stability
The reagent is stable up to the end of its
labeled expiration date, if properly stored at 2-
8*C (refrigerator temperature), protected from
light and contamination is avoided. Do not
freeze the reagent! Discard if it is found to
contain particulate matter.
The standard is stable up to the end of the
labeled expiration date, if properly stored at 2-
8*C and contamination is avoided.
Manual Procedure
1. Pipet into cuvettes the following volumes
(mL) and mix well.
Standard (S) Sample (U)
Reagent 1.5 or 1500ul 1.5 or 1500ul
Standard 0.050 or 50ul -
Sample - 0.050 or 50ul
2. Incubate all the tubes at 100ºC for 3 minutes.
3. Immerse test tube in room temperature water
for 2 minutes to cool, read absorbance of each
tube against Water Blank (Zero Absorbance)
at 600nm.
Formula for Glucose Concentration
C(mg/dL) = A sample/A standard x value of
standard (100mg/dL)
DELA CRUZ, KJ 2
 CCHM321 LABORATORY MIDTERMS

C(mmol/L) = A sample/A standard x value of

standard (100mg/dL) x 0.0556 (conversion
factor)
Normal Values (Profame)
Serum or Plasma (Fasting): 65-110mg/dL
Spinal Fluid: 40-70mg/dL
Clinical Significance
Diagnosis if the patient has Diabetes Mellitus
which is a disease that is characterized by
insufficient secretion of insulin from the
pancreas.
Post-Analytical Phase
Data and Results
Name, Age, Gender, Date of Birth,
Absorbance of unknown, and Absorbance of
standard.
Computation
C= Au/As x 100
Interpretation
Above reference range, below reference range
or within reference range.

DELA CRUZ, KJ 3