CLINICAL CHEMISTRY 1

GLUCOSE METHODOLOGIES
MRS.ANNABEL LARANJO
NOVEMBER 7, 2022

OBJECTIVES 2 Choice of method ● The ability of the

● To determine the specimens that can be used for method to detect
measuring glucose glucose in samples will
● To discuss the different methods of measuring glucose vary depending on the
including its principles, reagents used, and products method used
measured
● To know the preferred method of glucose measurement 3 Issues related to ● Ability of the test to rule
specificity out the presence of
GENERAL CONSIDERATIONS
glucose
● Assessment of the state of carbohydrate production, ● If you use that method,
metabolism, and excretion primarily involves the you will be definitely
measurement of glucose in body fluids sure that if it is negative
● The normal blood glucose level is approximately 70-110 or undetected, this
mg/dL depending on the assay method means that glucose is
● Variations in glucose metabolism result in levels as low as not found in that
20-30 mg/dL in hypoglycemia or as high as 800 mg/dL in substance or specimen
diabetes mellitus
● Therefore, any method used to quantitate glucose in 4 Ease of measurement ● Easy to measure
serum or plasma must be able to measure this material methods
accurately throughout a wide range or values ● Methods that employ
● Levels of CSF are normally about 40-70 mg/dL procedures that are
● CSF glucose concentrations are approximately 60-75% of easy to perform along
the concentrations in the blood of specific individual with its availability and
● When the level of blood glucose changes, approximately 2 is cost effective
hours is required to restore the CSF/blood glucose
equilibrium QUANTITATION OF BLOOD GLUCOSE
○ Remember: the brain cannot store nor synthesize
1 CHOICE OF SAMPLE
glucose. A decrease in the amount of glucose is
detrimental to the function of the brain or the ● Whole blood, Serum, Pleural fluid, Plasma, CSF, Urine
CSF. - Depending on the diseases you are considering
● Urine glucose is rarely quantitated in a patient and you want to determine the
○ A random specimen is usually screened in a presence of glucose in that specimen
qualitative fashion ● Standard clinical specimen (usually used) – venous
○ These screening processes should be able to collected specimen (can be plasma/serum)
detect urine glucose at levels of 30 mg/dL or ● Red blood cells have enzymes necessary to metabolize
higher glucose
○ In normal individuals, glucose should not be seen ● Separate the cells (e.g RBC, WBC) from the liquid portion
in urine. Glucose is an important substance in the of the blood (such as the serum/plasma) as soon as
body as a sole source of energy, thus they possible within 1 hour
shouldn’t be present in the urine in normal - There are cells present in the sample that will
individuals utilize glucose in the process
- To prevent substantial glucose loss by the cellular
QUANTITATION
fraction (e.g. WBC, RBC), they should be
separated or removed within 1 hr. This is
1 Choice of sample ● What sample is ideal for
especially applicable in cases wherein the WBC
quantitating blood
count is elevated (e.g. infection, hematologic
glucose and other body
malignancies leading to an increased WBC
fluids in order for you to
count such as in leukemia, etc)
diagnose disease/s?
● Fasting Blood Glucose (FBG) should be obtained in the
● With the choice of the
morning after an approximately 8-10 hour fast (should not
sample, it should be
be >16 hours) *diurnal variation*
properly extracted,
○ It should not be more than 16 hours. This is to
transported and
make up for the diurnal variation in the blood
processed
glucose.

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○ Fasting should be a maximum of 10 hrs, not less

Plasma Specimen ● Na Fluoride: gray capped
than 8 hrs.
(2mg/ml)- preservative should
○ Fasting more than the required time is already
be added
an invalid result. Inform the physician during the
○ Acceptable delay up to
test that patient overfasted, so the physician can
90 minutes without
make a decision whether to go with the
bacterial
procedure or repeat the test
contamination/leukocy
● CSF and urine can also be analyzed
tosis
○ In cases where you’re possibly considering
○ Used as a
infection in the brain such as meningitis (e.g. if it
preservative for
is bacterial or viral), the presence of glucose
glucose in 1.5 hrs
should be determined.
without bacterial
○ If glucose is lower than normal, most likely you
contamination
have a possible bacterial cause of meningitis.
● Fluoride or iodoacetate is
○ If CSF glucose is normal or high, most likely it’s
sometimes used in sample
fungal or viral meningitis.
collection since they inhibit the
○ Glucose in urine is important in considering
glycolytic process and
possible outflow of glucose from the blood into
prevent most glucose
the urine such as in cases of overt diabetes
consumption by the
mellitus
erythrocytes or leukocytes
● Urine glucose measurement is for monitoring purposes
● When refrigerated, serum/
ONLY
plasma glucose remains stable
○ Can also be used as a screening test for
for 48 hours but ideally it should
possible diabetes mellitus but it is not a
be a fresh specimen
diagnostic criteria. In the previous lecture, we
have different criteria in diagnosing diabetes
Whole Blood ● Glucose concentration in whole
mellitus
blood is approximately 11%
● After separation of either plasma or serum from the cells,
lower than in serum or plasma
glucose remains stable at room temperature for several
● % difference may vary with the
hours in serum samples
hematocrit of patient
● Glucose is less stable in plasma (as compared to that of
○ e.g anemic px where
serum, making serum as the ideal specimen for the
there is a decreased
venous collected blood in determining blood glucose )
hematocrit. The
● Glucose metabolism occurs in unprocessed specimens in
presence of anemia,
the ff. conditions:
particularly iron
○ At RT, its rate of metabolism is 7mg/dl/hr
deficiency anemia,
- Per hour, you lose 7 mg of glucose per
can impair glucose
100 mL of blood
homeostasis, and can
○ At 40 C, loss of approximately 2m/dl/hr
have a negative effect
○ In bacterial contamination, rate of metabolism is
on the glycemic
higher because of the bacteria that use glucose
control & can
(glucose is falsely low)
predispose to more
○ If you compare, there will be higher loss of
complications in
glucose or there is increase in glucose
diabetic patients
metabolism at RT compared to a lower temp
○ Therefore, you have to
take note of anemia or
Serum Specimen ● The use of collection tubes correct the anemia in
containing a gel-like material diabetic patients in
as a separation barrier between order to improve
the cells minimizes the loss of control of diabetes as
glucose through cellular well as when you have
metabolism anemia as a
○ The tube would complication of
separate the liquid diabetes, you have to
portion from the correct anemia in
cellular portion with order to have a
the use of gel matrix glycemic control.

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Capillary Blood ● using Hemo Glucometer METHOD OF GLUCOSE MEASUREMENT

○ Hemo Glucometer
measure blood
glucose
○ HGT- Hemoglucotest
● Increase in postprandial
specimens +/- 5 compared to
fasting venous sample
○ When you are using
postprandial
specimens meaning
specimens taken after
eating (2 hours after
eating), it is said to
have elevated values
CHEMICAL METHODS
2 CHOICE OF METHOD I.A OXIDATION-REDUCTION METHODS
● SPM assay (Spectrophotometric) I.A.1 ALKALINE COPPER REDUCTION METHODS
● Enzymatic method
● Glucose oxidase (enzymatic method) Principle: In a hot alkaline solution, Cupric ions are reduced to
● Hexokinase (enzymatic method) Cu+ by glucose and other reducing sugars w/ the formation of
Cu2O. (red ppt)
3 ISSUES RELATED TO SPECIFICITY ● Other reducing sugars: Lactose, Fructose, Pentoses
● Because specificity is important for considering a method
False High ● Blood taken after meal
for determining glucose
● Not clear PFF
4 EASE OF MEASUREMENT ● Delayed filtrate preparation of
sample
● Ease of measurement of glucose substance in the
specimen False Low ● Boiling is done at a lesser period
● NOTE: Converting mg% glucose to mmol/L concentration: of time
use factor 0.055 ● Exposure of the solution to air
QUANTITATION OF BLOOD GLUCOSE during the heating process
○ There is not enough time
● When measuring glucose, the method used is based on its
in reducing cupric ions,
reduction property
thus leading to a false low
● NOTE: There is one primary consideration applicable to all
result
glucose reduction methods
● Glucose is not the only reducing substance found in the
blood, and therefore, in the process of removing the
proteins, the ff. other non-glucose reducing substances
must be removed (at the start you have to obtain a protein
free filtrate):
○ Creatine, creatinine, uric acid, glutathione,
ergothioneine
○ They are all reducing substances which react like
glucose if they are subjected to the same
reduction procedure
● In order to obtain True Glucose Values, a PFF must be
prepared which should only contain Glucose as a
measurable reducing substance
However, this method is not specific for glucose only.
The presence of a green or yellow ppt would mean increases of
reducing sugar and if it is an orange ppt, we have moderate amount
of reducing sugars or glucose and brick red ppt mean presence of
large amounts of reducing sugars such as glucose, lactose,
fructose, and pentose.
This method is not specific for glucose only.
However, this method is not specific for glucose only.

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A.1.1 FOLIN-WU METHOD SOURCES OF ERROR:

● Specific method under alkaline copper reduction.
● PFF is made to react w/ phosphomolybdate to produce
phosphomolybdenum blue
● This method lacks specificity since it measures
saccharoids like glutathione, ergothione, ascorbic
acid, uric acid, creatinine, and glucuronides
○ Presents the same reaction as with glucose
○ This explains the high reference value of this
method which is 80-120 mg/dl
○ Saccharoids are non-fermentable, non-reducing
substances, concentration varies from 10-30
mg%
PROCEDURE
1.
Make PFF from 2ml WB, 9.0ml dis. H2o, 8ml of N/12
HsSO4, 1ml of 10% sodium tungstate (Chocolate
brown color solution). Filter/centrifuge.
2. Put 2ml PFF, 2ml STD, 2ml water blank in 3 Folin Wu
tubes, respectively, add 2ml Alkaline copper tartrate
(source of cupric ion) to each tube. Place in boiling
water for 6 mins.
3. Cool in cold water bath to stop any chemical reaction
for 3 mins. Stops Cu2+ → Cu+
4. After cooling, add 2ml phosphomolybdic acid reagent
(color reagent). Then dilute to 25ml mark of Folin Wu
tube, mix by inversion 2x. Read spectrophotometrically
at 420 nm.
Note: STD conc. = 100 mg%
● PFF is added along with the alkaline copper tartrate. It
should reach the constricted area of the Folin Wu tubes.
● In the presence of heat, there will be reduction of
cuprous ions to cupric ions and further conversion is
inhibited by cooling the solution for 3 minutes. This is to
prevent reversion of the procedure.
○ Incomplete heating or extension of the heating
process are also sources of error.
○ Incomplete boiling would give us insufficient
cuprous ions to react with the phosphomolybdic
acid resulting to false low results.
○ Extension of the heating process will convert
more cupric ions to cuprous, therefore, would
lead to false positive or false high result
● Shaking should be avoided because there is easy
oxidation of cuprous ions to cupric ions in the presence
of air.
● After cooling, the end product or the cupric ions are
added with the color reagent — phosphomolybdic
acid, it turns to the designated end product which is the
phosphomolybdenum blue, which is now a stable end
product and is measured using the Folin Wu method.
The intensity of its color is directly proportional to the
amount of reducing substances in our blood.

REACTION PRINCIPLE:

Folin Wu Tubes
● has a constriction near the end
○ purpose of constriction: to avoid exposure to air
● designed to prevent the oxidation of cuprous ions to cupric
ions since they are readily oxidized in the presence of air
Glucose, which is a reducing substance, together with the other
reducing substances, and with the presence of cuprous ions in
the presence of heat is converted to cupric ions. Cupric ions are
measured with the addition of a color reagent—
phosphomolybdic acid to form the phosphomolybdenum blue
which is the end product being measured in this method.

A.1.2 NELSON-SOMOGYI METHOD

● uses Alkaline reduction method

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● this is the most accurate Redox (oxidation-reduction)
method and believed to be a measure of True Glucose
because of the saccharoid-free PFF
○ we do not say that this is the most accurate
glucose method, but this is the most accurate
among the redox methods
● the Barium sulfate formed acts as an adsorbent to which
saccharoids adhere
● in this method, the PFF is made to react with
arsenomolybdate forming arsenomolybdenum blue
● Normal Reference Value
○ 65-95 mg/dL in serum
○ 50-70 mg/dL in CSF
PROCEDURE
● Measure 1 ml WB, add 15 ml distilled H2O, 2 ml BaOH in
50 ml Erlenmeyer flask
● Allow to stand for 30 seconds with constant mixing.
● Add 2 ml ZnSO4, after two minutes. Filter (result: avocado
color).
● Add 2 ml PFF to the tube, add copper solution, place in
boiling water for 10 mins. Allow to cool in 2 mins.
● Add color reagent Arsenomolybdate, then stand for 2
mins., dilute to 25 ml volume, invert 2x.
● Read at 490 nm, use 19x105 mm cuvette
A.1.3 NEOCUPROINE METHOD BY BITTER AND MANNING
A.1.4 BENEDICT’S METHOD
● A modification of Folin-Wu method which uses a Bisulfite
Copper Solution
○ Modified procedure in order to improve specificity
but also considered an obsolete method
Folin wu is less specific compared to Nelson Somogyi method
since it allows other saccharoides or reducing substances to react
with the reagent
A.1.5 SHAEFFER-HARTMANN-SOMOGYI METHOD
I.A.2 ALKALINE-FERRIC REDUCTION METHODS
A.2.1 Hagedorn-Jensen Method
● This method is more or less the same of that of alkaline
copper reduction method with the reduction of ferric ions
to ferrous ions with the production of the end product
being measured.
I.B.1 CONDENSATION METHODS
1 CONDENSATION WITH AROMATIC AMINES
1.1 O-TOLUIDINE METHOD (GLUCONEER)
PRINCIPLE
a. Glucose in a PFF reacts with 1⁰ aromatic amines, in a hot
glacial HAc to produce a stable green color
(N-glycosylamine and Schiff's base) w/ an absorbance
maximum at 630 nm.
b. This is the most specific non-enzymatic method for
glucose determination.
c. O-toluidine condenses with the aldehyde group of
aldohexoses (glucose & galactose)
d. The hot acidic solution (Acetic Acid) is for enolization or
enol formation. So that, the naturally occurring D-glucose be
enolized to L-glucose (enol forms) which are the active
reducing forms.
e. Sources of error may come from an icteric serum and from
a patient under a galactose load or suffering from
galactosemia
DISADVANTAGES
1. Harsh chemicals and strong reaction conditions
2. Heat and concentrated acid produce hazardous
conditions and are very destructive to instrumentation
over long term
a. Health hazard because o- toluidine is now
classified a carcinogen.
2 CONDENSATION WITH PHENOLS
2.1 CONDENSATION WITH ANTHRONE
ENZYMATIC METHODS
● A higher degree of specificity can be achieved
● Hazardous conditions and chemicals used in colorimetric
procedures are avoided
○ Enzymatic methods measure true glucose, not
reducing compounds.
■ They are the most widely used methods
because they are simple and rapid to
perform, are easily automated, use
small sample volumes, and are highly
specific.
● Disadvantage of Enzymatic Methods:
○ Hemolyzed sample
■ (false low – further glycolysis of
glucose) with enzymatic methods
because contents from RBCs may
interfere with glucose and NADPH
accumulation.
A GLUCOSE OXIDASE METHOD
1 SAIFER- GERSTENFIELD
● Glucose is measured by the reaction with Glucose
Oxidase, in which Gluconic acid and Hydrogen
Peroxide are formed.
● Hydrogen Peroxide then reacts with an Oxygen acceptor
such as O-Toluidine or O-Dianizidine in a reaction
catalyzed by peroxidase to form a blue color.
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Glucose if converted to hydrogen peroxide and Gluconic acid in the

order to form iodine with the use of molybdate iodide and also
presence of glucose oxidase enzyme and your hydrogen peroxide
water plus acetaldehyde if you are using ethanol. This is to
in the presence of a chromogenic acceptor such as O-Toluidine or
prevent the reversion of reaction. and truly measure the end
O-Dianizidine with the help of peroxidase enzyme is converted
product of this method.
eventually into water and oxidized chromogen.
This the end colored of oxidized chromogen is colored blue

To further illustrate the principle of the test.

So, glucose in the presence of glucose oxidase enzyme is 3 DEXTROSTIX
converted to hydrogen peroxide and gluconic acid. Your hydrogen
peroxide is further converted with the help of an acceptor which is B HEXOKINASE (HK) METHOD
your O-Toluidine or O-Dianizidine, with the presence of peroxidase ● The most specific method for blood glucose
enzyme is converted eventually into water and oxidized ● More accurate than the glucose oxidase method
chromogen. because of the coupling reaction using
This is known as your Trinder Reaction, and the oxidized glucose-6-phosphate dehydrogenase (G6PD) is highly
chromogen is known as 3-methyl-2-benzothiazolinone hydrazone specific
or N.N-dimethylaniline. ● Generally accepted as the reference method
○ Gross hemolysis and extremely elevated
● The peroxidase reaction is less specific than the bilirubin may cause a false decrease in results
glucose oxidase reaction Principle:
○ Various reducing substances in serum such as
uric acid, ascorbic acid, bilirubin, and
glutathione acid, inhibit the reaction by
competing with the enzyme, thus causing
falsely low results
● Main advantage: low cost
● End color depends on the chromogen used
● False increase results from strong oxidizing substances
such as bleach
2 POLAROGRAPHIC

PRINCIPLE It is a coupled reaction so first glucose is made to react with ATP in

● Amount of oxygen consumed in the oxidation of
glucose to gluconic acid is measured using a
polarographic oxygen electrode.
○ Therefore interferences encountered in the
peroxidase step of the glucose oxidase method
can be eliminated
This method is said to be more specific as compared to the
glucose oxidase method.
● Peroxide formed must be eliminated to prevent
reaction from reversing
The formed hydrogen peroxide need to react with iodide in the
presence of molybdate to form iodine
Or hydrogen peroxide need to react with ethanol in the process
of catalase in order to form water and acetaldehyde
If you have observed, hydrogen peroxide is not part of
the end product of the method. It is eliminated from the reaction
by the addition of other reagents such as iodide and ethanol in
the presence of hexokinase and magnesium ions to form the G6PD
plus ADP. So, this is known as the phosphorylation reaction. This is
followed by the reduction (it is redox already in this part). So,
G-6-Phosphate plus NADP+ in the presence of G6P
dehydrogenase is reduced to 6-phosphogluconate plus NADPH
plus H+. The end product would be the
6-phosphogluconate,NADPH, and H+
B HEXOKINASE METHOD
● NADPH has a strong absorbance at 340 nm
○ The rate of appearance of NADPH can be
monitored by SPM and is directly proportional to
the concentration of glucose in the sample.
● HK phosphorylates mannose and fructose but these
sugars are not present in sufficiently high concentration in
serum to interfere.

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● May be performed on serum or EDTA/heparin/
fluoride/oxalate or citrated plasma as well as urine, CSF
and serous fluids.
C GLUCOSE METHODS
Dehydrogenase
● II.C.A. SPM assay
● II.C.B. Electric current assay
D CLINITEST
E TEST TO MEASURE FOR GLUCOSE
1 FASTING BLOOD SUGAR (FBS)
● The preferred method for dx diabetes
● 8-10 hour fast without any caloric intake
● FBS of >126 mg/dL on at least 2 different days is dx for DM
○ FBS: 110-125 mg/dL - impaired fasting glucose
● To detect any disorder of glucose metabolism, mainly
diabetes, and is used as an aid in glucose management
● Normal values:
○ Fasting serum= 70-100 mg/dL
○ Fasting whole blood = 60-100 mg/dL
2 RANDOM BLOOD SUGAR
● A screening test for diabetes but not diagnostic
● A check response of glucose level post meals
● To check the ability of the body to utilize glucose as a
source of energy after a certain period of time
● An RBS of 200 mg/dL indicates diabetes
● The test results must be confirmed on another day with
FBS or OGTT
● Polydipsia, polyuria and unexplained weight loss + RBS >=
200 mg/dL is presumptive of DM (diabetes mellitus)
● Patient preparation = None so far
● Normal values = 85-125 mg/dL
Aside from FBS and random blood glucose, you can also measure
the glucose tolerance of the individual.
TWO METHODS OF GLUCOSE TOLERANCE TESTS:
■ Pedia Px: 1.75 g/KBW (kilogram body
weight
● This test measures FBS before giving glucose load.
○ So, you have to get the baseline fasting blood
glucose before giving the glucose load
○ Other lab examines urine glucose simultaneously
● If FBS > 140 mg/dL, terminate the test
● If FBS < 130 mg/dL, administer the glucose load
● Collect (blood) samples every 30 minutes for 2 hours
○ (Other labs have these collection schedule: 30
mins, 1 hr, 2 hrs, and 3 hrs after giving
glucose load)
○ So, you have successive determination of blood
glucose
CRITERIA FOR DIAGNOSING DIABETES USING OGTT
● Diabetes is considered to be present if:
○ Fasting glucose is >140 mg/dl
● Impaired Glucose Tolerance is considered if: 2 hours
value 140-200 mg/dl and one other value >200 mg/dl
● Gestational diabetes is considered present if 2 or
more values exceed:
○ Fasting: 105 mg/dl
○ 1-hour: 190 mg/dl
○ 2 -hour: 165 mg/dl
○ 3- hour: 145 mg/dl
Procedure:
1. Obtain a fasting blood sample and urine is collected.
2. Glucose load dissolved in flavored water is administered
(AD) orally.
a. The drink should be ingested in approx. 5
min. and test is timed from the
commencement of the drinking
3. Blood and urine samples are then obtained by 30 mins, 1
hr, 2 hrs, and 3 hrs after ingestion of glucose load
a. The patient should remain seated throughout
the test

A Janney–Isaacson Method OGTT Schedule (4 hours)

(Oral Glucose Tolerance Test (OGTT)
BLOOD URINE
● Designed to evaluate the insulin’s response to a
physiologic glucose challenge FBS = 6:00 AM; 6:45 drink 6AM
● In diabetes, glucose piles up in the bloodstream,
especially after meals. If a glucose load is given to a 1st 30 mins = 7:15 1st
diabetic patient, the blood glucose rises higher and
returns to baseline more slowly than it normally does. 2nd 1:00 hr = 7:45 2nd
● Patient Preparation:
3rd 2:00 hr = 8:45 3rd
○ 3 days prior to the test, the patient is on a CHO
diet of 150 g/day
4th 3:00 hr = 9:45 4th
○ (Before the test) Patient must fast for 6-8 hours:
Some requires 8-14 hrs 5th 4:00 hr = 10:45 5th
○ Dose range of load to be given to the px: 50g;
(you also measure the urine simultaneously with blood glucose)
75g; and 100g
■ Adults: 75g of glucose
■ Pregnant women: 100g NORMAL VALUES

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Fasting Same as FBS normal values
- Fasting serum= 70-1000 mg/dL
- Fasting whole blood = 60-100 mg/dL
30 mins 30-60 mg/dl above fasting
60 mins 20-50 mg/dl above fasting
120 mins 5-15 mg/dl above fasting
180 mins fasting level and below
● Graph Showing the comparison between the normal
glucose levels in a diabetic patient (purple) from a
non-diabetic patient (blue)
- At the start, when the glucose load was given to the
non-diabetic patients, there will be a subsequent rise in
the glucose blood level
- 30 mins up to 1 hour from the giving of glucose load and
there will be an expected decline to normal values at 2
hours after giving the glucose load or after eating.
- It should be expected that 2 hours after eating the glucose
levels should return to normal as a result of insulin actions.
- However if you are diabetic, the glucose initially is very
high from the start which is above 100 mg/dl
- There is a rapid rise in the blood glucose immediately after
giving the glucose peaking 1 hour after giving the glucose
load and it reduces slowly
- However, it is usually above the normal level 2 hours after
the glucose load
- Usually, the value is usually above 150 mg/dl for diabetic
patients 2 hours post glucose load
- We can say that there is a problem with the insulin
production in diabetic patients
In diabetic patients, there is a gradual decrease in the glucose load
2 hours after giving the glucose load, or there is a derangement in
the rise and fall of the blood glucose level.
B Exton Rose or Divided Dose Method
(2-hour Postprandial Test)
● A test taken after a meal, is an excellent screening test for
diabetes
● Glucose concentration in a fasting blood specimen, obtained
2 hours after a meal is rarely elevated in normal persons but
is significantly increased in diabetic patients
● Patient preparation
1. Patient should be in a CHO diet for 2-3 days before
testing
2. Fasting from foods overnight, at least 12 hours is
required
Blood sample is collected after the 12-hour fast
3. After an overnight fast, the patient eats a high CHO
breakfast
○ The meal includes orange juice, cereal with
sugar; toast and milk
4. The patient should remain at rest during the 2 hour
interval. 2 hours after the patient finishes eating
breakfast, a venous blood sample of about 5 ml is
obtained
● Normal value: must be lower than 120 mg/dl. However it is
elevated for patients who are diabetic.
C Intravenous Glucose Tolerance Test
● May be performed instead of OGTT in patients with poor
absorption of orally administered (AD) glucose or the
inability to tolerate the oral CHO load
● Glucose load of 0.5g/kg body weight is administered
intravenously (IV), and glucose levels are determined
every 10 minutes for 1 hour
● Insulin levels are often requested with this test
OTHER TESTS
1 SELF-MONITORING OF BLOOD GLUCOSE (SMBG)
● American Diabetes Association (ADA) has recommended
that individuals w/ diabetes should monitor their blood
glucose levels in an effort to maintain levels as close to
normal as possible
● For type 1 DM, the recommendations are 3-4x/day
● For type 2 DM, optimal frequency is unknown (depending
on the availability of the strips and comfortability of
patients)
● Patients should be taught how to use control solutions
and calibrator to ensure accuracy of results
● Urine glucose should be replaced by SMBG. but urine
ketone testing will remain for type 1 and gestational
diabetes (since they are prone to ketosis/ketoacidosis)
2 GLYCOSYLATED (GLYCATED) HEMOGLOBIN
● Determines the long term blood glucose regulation
● Used to describe the formation of Hb compound formed
when glucose (a reducing sugar) reacts with the amino
group of Hb (a CHON) to form the glycosylated
hemoglobin
● Glucose attaches non-enzymatically to Hb to form
ketoamine
○ The rate of formation is directly proportional to
the plasma glucose concentration
○ Therefore if there are more glucose in your blood,
there will be more glucose that is attached in your
hemoglobin
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● Reflects the average blood glucose level over the
previous 2-3 months
○ This is the approximate red blood cell life span
which is 120 days
GLYCOSYLATED (GLYCATED) HEMOGLOBIN - HbA1C
● is the largest sub-fraction of normal HbA in both diabetic
and non-diabetic subjects
○ formed by the reaction of the B-Chain of HbA
with glucose
● most commonly detected glycosylated hemoglobin and
reliable method of monitoring long term diabetic control
than RBS (random blood sugar)
● Is a glucose molecule attached to one or both N-terminal
valines of the B-polypeptide chains of normal adult Hb
● Normal value: 4.5-8.0 mg/dL
● Levels determined by 2 factors
1. Average glucose concentration and
2. RBC life span
● If the RBC life span is decreased
because of another disease state such
as hemoglobinopathies, the Hb will have
less time to be glycosylated and
glycosylated hemoglobin will be lower
METHODS OF MEASUREMENT
1 Based on the charge differences between glycosylated Hb
(GHb) and non-glycosylated Hb (non-GHb)
● cation exchange chromatography,
electrophoresis, and isoelectric focusing
2 Structural characteristics of glycogroups on Hb
● affinity chromatography and immunoassay
● Affinity chromatography
○ Preferred method
○ Glycosylated Hb attaches to the
boronate group of resin and is
selectively eluted from the resin bed
using a buffer
○ Not temperature dependent
○ Not affected by hemoglobin F, S, or C
3 FRUCTOSAMINE
● Refers to glycosylated albumin and other CHONs
● Forms a ketoamine linkage between the glucose and
CHON
● Reflects blood glucose levels for 2-3 weeks before
sampling
○ Serum albumin has a half-life of 2-3 weeks
● Analytical Procedures:
1. Column Chromatography
2. Affinity Chromatography
3. Colorimetric Assays
a. If frustosamines are reacted with a strong
acid, a cyclic derivative known as
5-hydroxymethylfurfuraldehyde forms
b. In alkaline solution, fructosamine reduces
nitro-blue tetrazolium
4 KETONES
● Are products of incomplete fat metabolism
(β-oxidation)
○ include acetone (2%), acetoacetic acid (20%),
3-β-hydroxybutyric acid (78%)
● Produced by the liver through metabolism of fatty acid
(FA) to provide a ready energy source from stored lipids at
a time of low carbohydrate (CHO) availability
● Low level is present in the body at ALL times but levels
can be elevated to meet the energy needs in cases of
CHO deprivation or decreased CHO use
○ Such as DM, starvation/fasting, high-fat diets,
prolonged vomiting and glycogen storage
diseases
● Major problems resulting from excessive ketones:
○ Acidosis
○ Electrolyte Loss
● Ketonemia is the presence of ketone in the blood while
ketonuria is the appearance of ketone bodies in the urine
● Measurement of ketones is recommended for patients
with:
○ Type 1 DM during acute illness
○ Stress
○ Pregnancy
○ Blood glucose > 300 mg/dL
○ Sign of acidosis (such as rapid deep breathing
aka the Kussmaul sign of respiration)
● Specimen is fresh serum or urine -> must be tightly
stoppered & analyzed immediately
● No method reacts with all ketone bodies
5 MICROALBUMINURIA/URINE MICRO ALBUMIN TEST
● DM causes progressive changes to the kidneys and
ultimately results in diabetic renal nephropathy
● Presence of albumin in urine is an early sign of
nephropathy
● Microalbumin measurements are useful to assist in
diagnosis at an early stage and before the development of
proteinuria
● Annual assessment of kidney function is recommended
for diabetic patients
● Microalbumin concentrations are between 20 mg/day to
300 mg/day;
● Proteinuria: > 0.5 grams/day
Microalbuminuria Defined as persistent albuminuria in 2
out of 3 urine collections of 30-300
mg/24hr, 20 - 200 ug/min, or
Albumin-creatinine ratio of 30-300
ug/mg creatinine
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CC 1: GLUCOSE METHODOLOGIES

Clinical proteinuria Albumin-creatinine ratio of ≥ 300

or Macroalbuminuria mg/24hrs,
> 200 ug/min, or
≥ 300 ug/mg creatinine

6 ISLET AUTOANTIBODY, INSULIN, AND C-PEPTIDE

TESTING
ISLET ANTIBODY TESTING
● Presence of autoAb to the B-islet cells of the pancreas is
characteristic of type 1 DM
● Not recommended for routine screening, but might
identify patients at-risk and prediabetic patients
INSULIN MEASUREMENT
● Are not required for the dx of DM
● However, in certain hypoglycemic states (e.g., those with
insulinoma or excessive production of insulin due to a
tumor in the pancreas), it is important to know the
concentration of insulin in relation to the plasma glucose
concentration
C-PEPTIDE ASSAY
● Useful when inappropriate insulin administration (AD)
must be distinguished from endogenous insulin
production
● Hypoglycemia in a diabetic patient with access to insulin
may be due to excessive use or due to the endogenous
insulin
● Exogenous insulin has been purified and C-peptide has
been removed
● If insulin concentration are greater than the C-peptide
concentration, the plasma insulin is from exogenous
administration
● Therefore, if you measure the C-peptide you will be able to
determine the insulin concentration in the body is from the
exogenous or endogenous source

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