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27 Diabetes Mellitus: TheRole oftheLaboratory
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403
Postanalytical Aspects
As pointed out in section “Fasting blood glucose,” basal and
afterload blood glucose values during OGTT should be
expressed in mmol/L. However, many laboratories still
express blood glucose in mg/dL.Plasma glucose reading 2h
after OGTT of less than 7.8mmol/L (140mg/dL) is considered normal. If the value obtained is between 7.8 and
11.0mmol/L (respectively 140 and 199mg/dL), the diagnosis of IGT is made.
Diabetes mellitus is diagnosed (with a second diagnostic
criterion met) if blood glucose 2h after OGTT is greater than
or equal to 11.1mmol/L (200mg/dL).
Blood Glucose Self-Monitoring Using POCT
According to the most recent position statement of the
American Diabetes Association, self-monitoring of blood
glucose using POCT is considered an essential component in
the management of the diabetic patient since it helps monitor
glycemic control, detects hypoglycemia, assists in adjusting
therapy, helps in understanding glycemic uctuations, and
can reduce or delay the onset of vascular, neurological, renal,
and retinal complications.
Self-monitoring of blood glucose is carried out by sophisticated electronic devices that are easy to use and take up
little space. There are different types of instruments on the
market based on different principles.
A small blood sample is taken from the diabetic patient by
supercial skin puncture, usually on the outside of the ngers or, if preferred, on the ngertip, the sites with the best
blood supply. Alternative sampling sites (forearm or ear
lobe) may also be used, especially in children. The drop of
capillary blood (a few microliters) is then deposited on a
strip inserted into the machine’s slot.
The principle on which the most common glucose meters
are based is the quantication of an enzymatic reaction, the
oxidation of glucose, which is proportional to the concentration in the blood. The reaction is carried out by placing a
blood sample on a strip containing glucose oxidase (GOD)
as the enzyme. During the oxidation reaction, gluconic acid
and H2O2 are produced. Subsequent quantication of the
reaction can be obtained either reectometrically (measurement of light reected from the colored test strip as a result
of the chromogenic reaction catalyzed by glucose oxidase
and peroxidase and subsequent conversion of the signal to
glucose concentration) or impedance (electrical conductivity
of blood induced by the electrical current generated by glucose oxidation). The electrons produced by the reaction form
a current that is specically calibrated to reect the glucose
concentration in whole blood.
In addition to devices based on the GOD reaction, there
are others commercially available that use the reaction of
glucose-dehydrogenase (GDH), an enzyme that catalyzes
the formation of glucono-δ-lactone d NADH.Three different
types of enzymes can be used in GDH-based glucose meters:
glucose 1-dehydrogenase (1-GDH), glucose-6-phosphate
1-dehydrogenase (G6PDH), and quinoprotein glucose dehydrogenase (PQQ-GDH). As with all enzymes belonging to
the class of dehydrogenases, they can have the natural cofactor (NAD or NADP) unbound or weakly bound to the enzyme
(1-GDH and G6PDH) and therefore must necessarily be
added to the glucose meter test strip or strongly bound to the
enzyme as in the case of PQQ-GDH.
The heterogeneity of the devices, the modalities of use,
and the related analytical techniques make necessary the
training of the patient in the use of the glucometer (with particular attention to the preanalytical, analytical, and postanalytical aspects) and the periodic verication of the training.
The choice of the device must be made based on a series of
considerations, of which the economic aspect is only one of
the points to be considered.
Preanalytical Aspects
Many preanalytical factors can affect the accuracy of blood
glucose measurement, including patient preparation, storage,
and use of strips. Preanalytical procedures are elementary
but can be a source of important measurement errors, so,
even if trivial, they must be explained very well to the patient.
Before proceeding to the puncture site, a thorough cleansing
of the skin is necessary, especially if foods with a high sugar
content have been handled. The skin can be disinfected with
alcohol, taking care that no residue remains that could hemolyze or dilute the drop of blood obtained. The choice of the
type of lancing device (with a larger or smaller needle gauge)
must be made by taking into account the skin thickness of the
subject, who must undergo self-monitoring.
Other patient-related preanalytical factors, such as diseases restricting circulation, excessive hydration, or extreme
hematocrit levels, may make the blood glucose measurement
unreliable.
Test strips should be stored correctly in their original containers and carefully resealed after each use. They should not
be exposed to high temperatures, humidity, altitude, or other
environmental factors that can interfere with chemical reactions. In addition to the strips, the instrument should be
maintained in good condition and serviced periodically.
Analytical Aspects
Although self-monitoring of blood glucose (SMBG) devices
have traditionally been held to less stringent analytical standards than laboratory tests, there are guidelines that dene
analytical goals. To meet analytical accuracy requirements,
95% of capillary measurements must be within ±15mg/dL
(±0.83mmol/L) of the mean of the values determined by the
reference procedure for glucose concentrations below
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A. Mosca and M. Montagnana
100 mg/dL (<5.5 mmol/L) or ± 15% for values above
100 mg/dL. However, not all available instrumentation
achieved the same analytical standard.
The patient undergoing self-monitoring must implement
specic procedures to ensure proper meter operation and
quality of results. It is essential that calibration be performed
for those meters that do not have a self-calibration feature.
The calibration is a procedure by which you put the signal
provided by the instrument (instrumental reading) with the
quantity to be measured and correct the differences between
different batches of strips. It can be done by inserting a code,
chip, or calibrating strip, which is different from instrument
to instrument. It improves accuracy, so it is a process that
must be repeated over time. Calibration errors are a persistent cause of error.
Control materials, consisting of control strips or solutions
of known concentration simulating the analytical signal
detected by the system reader, shall be used to verify the
quality of the data produced. This must be carried out when
the instrument is rst used, and then a new lot of strips are
opened if the device is damaged or if there is any doubt about
the reliability of the data.
It is expected that a periodic check for correlation or interchangeability with the central laboratory results will also be
implemented on the POCT analyzers, which serves both to
check that the dened comparability requirements between
the two systems are maintained and to transfer the documented quality of the laboratory analyzer to the POCT
system.
There are many substances (e.g., drugs) that can interfere
with the chemical reactions taking place on the test strip. The
most important of these are ascorbic acid, paracetamol, and
maltose.
Very high plasma triglyceride concentrations, typically
above 2000 mg/dL, can lead to an underestimation of
blood glucose by a glucometer because they decrease the
amount of glucose present in the capillary volume. High
bilirubin concentrations (usually greater than 500mg/dL),
as seen in jaundiced infants, patients with hepatopathy,
hepatitis, or some forms of anemia, can create interference, especially when using glucose meters based on the
GDH method.
Ascorbic acid, commonly known as vitamin C, has the
potential to interfere with glucometer measurements,
although levels of 1–2mg/dL do not generally result in signicant interference. However, overuse of vitamin C can
cause this type of interference.
Finally, the type of interference may depend on the chemistry of each test strip, so it may also vary from one meter to
another.
As new glucose meters continue to appear on the market,
it is helpful to continually consult the literature to see comparative assessments.
Postanalytical Aspects
Two main postanalytical issues need to be known: the international unit of measurement (mmol/L) choice and the use
of plasma-calibrated strips. In 2001, the International
Federation of Clinical Chemistry and Laboratory Medicine
(IFCC) recommended using plasma-calibrated strips,
which transform the value obtained from whole blood into
the value obtained from plasma. This allows the data
obtained by the capillary blood glucose meter to be compared with the data obtained by measuring blood glucose in
venous plasma.
Glycemic Variability
Various devices that continuously measure blood glucose in
the subcutaneous tissue, known as continuous glucose monitoring (CGM), have recently become available. The availability of these devices now makes it possible to obtain a
precise prole of a single patient’s glycemic variations for
some days (up to 14 days) and to precisely quantify this
quantity. It has been known for a long time that, with the
same glycemic control, subjects with higher peaks of glycemic uctuations, more frequently, are at greater risk of complications than others.
The advantages of using these systems compared to
traditional POCT for SMBG are unclear. However, it
seems, based on the results obtained in a very recent clinical trial, that the use of CGM systems, compared to
SMBG systems, resulted in an increase in the time spent
in euglycemia, a reduction in the number of severe hypoglycemias in patients with type 1 diabetes, and a loss of
sensitivity to hypoglycemia. This study, therefore, and
the others that will undoubtedly follow, would seem to
support using such tools, at least in a population of highrisk diabetics.
Ketone Bodies
Measurement of blood (ketonemia) or urine (ketonuria) concentrations of ketone bodies (acetone, acetoacetate, and -βhydroxybutyrate) is recommended to aid in the diagnosis of
diabetic ketoacidosis in patients with type 1 diabetes and
gestational diabetes. Indeed, the presence of ketones may
indicate the onset of ketoacidosis, a condition that requires
immediate medical action. In the patient with this condition,
the increased concentration of ketone bodies leads to a lowering of the blood pH, resulting in acidosis (arterial pH <7.3
and/or HCO-3 levels <15mEq/L).
Under normal conditions and with a balanced diet, ketone
bodies are produced in small amounts because acetyl-CoA is
mainly used in the citric acid cycle. Conversely, under condi-
27 Diabetes Mellitus: TheRole oftheLaboratory
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tions (e.g., diabetes and prolonged fasting) that lead to accumulation of acetyl-CoA (as a result of increased
gluconeogenesis in the liver and fatty acid oxidation in muscle and liver), the liver gets rid of excess fatty acids by producing ketone bodies, which it sends through the bloodstream
to peripheral tissues where they are oxidized to produce
energy (hydroxybutyrate).
The symptoms that characterize diabetic ketoacidosis are
fatigue, general malaise, polyuria, polydipsia, cardiac
arrhythmias, drowsiness, weight loss, and bradypnea. Other
signs that appear are dehydration, hypotension, ECG abnormalities, brain dysfunction, loss of muscle mass, and
Kussmaul’s breath.
The concentration of ketone bodies can be measured
both in plasma and in urine. However, we must emphasize
that ketonuria does not express exactly the level of ketonemia in real time, but it provides indirect information about
it, with a delay proportionate to the length of the interval
between emptying the bladder and the other. Moreover, it
does not detect the presence of all three ketone bodies but
only of acetoacetate since β-hydroxybutyrate is not detectable by standard sticks, and acetone is eliminated by
breath.
Preanalytical Aspects
Because acetone is volatile and acetic acid may be an energy
source for bacteria, the analysis of fresh urine samples is
recommended.
Increased levels of ketone bodies may be present in
healthy subjects during fasting and in the morning urine of
30% of pregnant women.
Analytical Aspects
Urinary tests determine the presence of acetoacetate
(which accounts for 10–25% of the total ketone bodies)
using a colorimetric reaction with sodium nitroprusside.
At alkaline pH, sodium nitroprusside reacts with acetone
and acetoacetic acid (a product of acetone), forming a
complex that results in color development. It is susceptible
to acetoacetic acid and does not react with more complex
ketone bodies.
False positives (in the case of the intake of drugs based on
sulfhydryl compounds such as captopril, acetylcysteine, and
penicillamine) and false negatives may occur, the latter
related to an excess of vitamin C, excessive intake of liquids
that can dilute the urine, and the degradation of the test strips
over time.
In recent years, POCT methods have been developed for
determining ketone bodies in capillary blood. These instruments measure β-hydroxybutyrate, which represents the predominant and earliest-appearing ketone.
405
Albuminuria
Urinary albumin is an essential biomarker for the development of diabetic nephropathy and is also a known marker of
endothelial damage, which leads to a signicant risk of cardiovascular disease. Therefore, its determination is indicated
in diabetic patients at risk of renal (renal failure) and cardiovascular complications.
The progression of urinary albumin excretion increases
the likelihood of developing chronic renal failure. In addition, the presence of subclinical amounts of albumin in the
urine (previously identied by the term “microalbuminuria,”
to be dropped) is considered a cardiovascular risk factor for
diabetic and nondiabetic patients.
Even after the institution of the treatment plan, measurement of albuminuria is recommended to evaluate the effectiveness of treatment. Guidelines indicate that urine albumin
measurement should be performed annually in patients with
type 1 diabetes with a disease duration greater than 5years,
all patients with type 2 diabetes regardless of disease duration, and pregnant patients. For the diagnosis of diabetic
nephropathy, albuminuria must be positive in at least two out
of three samples collected within 3–6months.
In the DCCT and UKPDS trials, it was found that intensive therapy for diabetes reduces the risk of increased urinary
albumin excretion (UAE) and overt nephropathy in diabetic
patients. A new guideline on managing chronic kidney disease, which also covers the use of albuminuria measurement,
has recently been published and is referred to for further
discussion.
Preanalytical Aspects
The standardization of urinary albumin measurement is not
optimal, so making clear and consistent recommendations is
essential. Urinary albumin excretion can be increased by
physiological factors such as exercise, posture, and diuresis,
and samples should not be collected after strenuous exercise
(up to 2–3days before testing), in the presence of a urinary
infection, during a period of convalescence, or after surgery.
The urine sample from the 24-h collection and the rstmorning specimen (on which to measure albumin and creatinine) are suitable. The collection of the rst-morning sample
is preferable because it is less affected by biological variability than the other methods listed.
Due to the high individual and daily variability, performing at least three different urine collections on three different
days is advisable. The urine sample should then be stored at
4°C (stable for up to 2weeks) or at −80°C if more extended
storage is required (stable for up to 5months). Freezing at
−20°C is not recommended. Freezing the sample may lead
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A. Mosca and M. Montagnana
to a decrease in albumin. To avoid this problem, the sample
should be thoroughly mixed before analysis. Visually turbid
samples should be centrifuged to remove precipitates and
cellular components. Possible bacterial contamination and
glucose do not affect the investigation.
Analytical Aspects
Both semi-quantitative and quantitative tests are available
for the determination of increased UAE.Semi-quantitative
tests are recommended only in screening programs because
they have low sensitivity since they cannot detect modest
increases in urinary albumin excretion, which characterize
the early stages of diabetic nephropathy. In contrast, quantitative assays are highly sensitive.
Several methods have been developed for measuring
albumin in urine, including radioimmunoassay, enzymelinked immunosorbent assay (ELISA), radial immunodiffusion, immunoturbidimetry, and nephelometry. Each method
has advantages and disadvantages, so the choice depends on
the laboratory. In general, however, these assays have similar
inaccuracies, limitations, and reference ranges. In any case,
the positivity for urinary proteins with the test strips must be
conrmed by laboratory measurements. Regarding the measurement method for albumin, the recommendations for
immunochemical techniques are a coefcient of variation
(CV) <15% and a detection limit of approximately
2mg/L.Based on observational clinical studies, other indications suggest a more stringent value for imprecision: CV
<13% for albumin and CV <6% for the albumin/creatinine
ratio.
Postanalytical Aspects
The reference intervals for making a diagnosis of diabetic
nephropathy differ by type of sample collection and are
shown in Table27.2.
Units can be in mg/24 h for 24-h excretion or in mg/g
creatinine, which gives the same result. The use of mg/mmol
Table 27.2 Reference ranges of urinary albumin
Urinary excretion of albumin
Sample type and
measurement
units
First sample of the morning
mg/g creatinine
mg/mmol
creatinine
24-h urine collection
mg/24h <30 30–300 >300
Physiological or
mildly increased
(class A1)
<30
<3
Moderately
increased
(class A2)
30–300
3–30
Severely
increased
(class A3)
>300
>30
creatinine generates a different numerical value. The suggestion for deciding which unit to use is to establish uniformity
of reporting among laboratories in the same geographic area.
It is strongly discouraged to report albumin in mg/L.
There are no different decision levels for the sex of the
diabetic patient, while for the albuminuria/cardiovascular
risk association, the decision levels are still under study, but
epidemiological studies suggest a lower concentration than
that decided for diabetic nephropathy.
Glycated Hemoglobin
The red blood cells of a healthy adult subject contain mainly
hemoglobin A (HbA) (97%), hemoglobin A2 (HbA2)
(2–3%), and traces of fetal hemoglobin (HbF) (0.5–1%).
Chromatographic analysis of HbA led to the identication
of several minor fractions: HbA1a, HbA1b, and HbA1c, collectively referred to as HbA1. HbA1c is a signicant fraction
and constitutes about 80% of HbA1. HbA1c is the product of
the nonenzymatic condensation reaction between the aldehyde group of glucose and the amino residues of some
amino acids, mainly the terminal valines, of the hemoglobin
β-chain.
Because red blood cells are permeable to glucose, the
extent of HbA1c formation is directly proportional to the concentration of glucose to which red blood cells are exposed in
the circulation and the relative exposure time.
The measurement of HbA1c in the blood is the gold standard for assessing glycometabolic control in diabetic subjects. From the results of the DCCT study, elevated HbA1c
levels increase the risk of micro- and macrovascular complications; consequently, this parameter is used as a target for
possible therapeutic choices. Moreover, HbA1c measurement
is also used to conrm the diagnosis of diabetes: values
≥48mmol/mol (6.5%), if conrmed on at least two subsequent occasions, would allow the diagnosis of diabetes to be
made. In any case, it is not advisable to use HbA1c for diagnosis in children and adolescents, in women within 2 months
after childbirth, in subjects treated with glucocorticoids,
immediately after surgery, or in the presence of acute
pancreatitis.
The frequency of HbA1c measurements is theoretically
related to the average life of red blood cells (4months) and,
therefore, should be three times per year. The ADA recommends measuring HbA1c twice a year in patients in stable
metabolic control who have reached the therapeutic targets,
with a greater number of determinations for patients in
unstable control. Finally, in patients with gestational diabetes and in particular cases, measurements can also be made at
shorter time intervals. In any case, the measurement carried
out less than 2 months later is considered inappropriate for
evaluating glycemic compensation.
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407
Preanalytical Aspects
Table 27.3 shows the main interferences in the measurement
of HbA1c, specically listed regarding the risk of false positives or negatives using this parameter for the diagnosis of
diabetes. There is no doubt that there are differences between
males and females in HbA1c values in the absence of diabetes, with females showing slightly lower levels than males.
Age appears controversial, but some authors reported an
increase in HbA1c of about 1mmol/mol (0.1%) from age 30
to 70for each decade of age starting at age 30.
Regarding ethnicity, it has now been ascertained that
black subjects have, on average, higher HbA1c values than
Caucasians, with the same glycemic control, although the
risk of developing microvascular complications in the
long term does not seem to be affected. In the population,
there are also subjects with a “fast” or “slow” glycation
phenotype, depending on whether their HbA1c values are
higher or lower than average, respectively, with the same
average daily glycemia. Other factors, such as lifestyle
and diet between summer and winter, are related to possible seasonal variations in HbA1c concentrations. Such
variations can cause changes of 5–7% from the mean
annual value.
Table 27.3 Limitations in the use of glycated hemoglobin for diagnosing diabetes
Factors Risk in the diagnosis
Analytical interferences (potentially eliminable depending on the
analytical method)
Hyperbilirubinemia Underestimate
Hypertriglyceridemia Overestimate
Increase in white blood cells Overestimate
Presence of hemoglobin variants Over- or
underestimate
Physiological factors (potentially known before analysis)
Pregnancy Underestimate
Age Underestimate
Sex Over- or
underestimate
Seasonal variations Over- or
underestimate
Genetic determinants Over- or
underestimate
Presence of hemoglobin variants Over- or
underestimate
Pathological factors
Type 1 diabetes of recent origin Underestimate
Hemolytic anemias Underestimate
Iron deciency anemia Overestimate
Recent blood loss, transfusions Underestimate
Splenectomy Overestimate
Kidney failure Underestimate
Anti-retroviral therapy Overestimate
Erythropoietin therapy Underestimate
Alcohol abuse Underestimate
Malaria Underestimate
The intake of vitamin C or E seems to reduce HbA1c
due to an inhibition of the glycation process.
Hypertriglyceridemia, hyperbilirubinemia, chronic salicylate intake, and opioid dependence may cause interference
with the method, resulting in increased HbA1c values.
Alcohol consumption also inuences HbA1c levels since it
has been shown that abstinence from alcohol consumption
in alcoholics in rehabilitation is associated with a decrease
in HbA1c of about 4mmol/mol (0.4%). Finally, the presence of hemoglobin variants can cause positive and negative interference, depending on the method in use.
For sample collection, venous or capillary blood samples
can be taken using a lancing device. The anticoagulant generally used is EDTA.The stability of the whole blood sample
is 3days at 20°C, at least 5days at 4°C, and at least 6months
at −80 °C.If samples are stored at −80°C, they should be
tested within a short period of time after thawing. Some
diagnostic kit manufacturers have introduced capillary blood
collection systems that provide stability of approximately
1–2weeks at room temperature. However, these systems are
method dependent and cannot be adapted to other analytical
systems.
Analytical Aspects
To date, more than 100 different methods have been used to
measure HbA1c. These methods can be grouped as follows:
chromatographic methods based on the isoelectric point difference between HbA1c and HbA (ion exchange, highperformance liquid chromatography [HPLC]) or the presence
of glucose covalently linked to hemoglobin (afnity chromatography); immunochemical and enzymatic methods (detection of ketoamine). Usually, the results obtained by different
methods are very well correlated, and there is no evidence
that data obtained by one method are superior to data
obtained by another.
It is important to remember that the HbA1c result should
always be contextualized; if it is in contrast with the patient’s
clinical picture, the test should be repeated, using a different
method (if possible) or assessing glycemic control using
other analytes (e.g., glycated albumin [GA]).
As with other tests, the measurements must be standardized for the HbA1c gure to be usable. To this end, the IFCC
established a working group in 1995 to standardize HbA1c
results globally. Subsequently, in 2007, another working
group consisting of members of the ADA, the European
Association for the Study of Diabetes, and the International
Diabetes Federation (IDF), based on the IFCC’s achievements, produced a consensus document on HbA1c standardization, which dictated the strategy at the global level.
In the National Glycohemoglobin Standardization
Program certied system, HbA1c is reported as a percentage
of total Hb. In contrast, the IFCC has recommended that
HbA1c be expressed in mmol/mol (HbA1c/Hb). The compari-
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A. Mosca and M. Montagnana
son between the two systems has created an equation allowing conversion between the two units. In order to ensure a
standardized result, it is considered appropriate that the inaccuracy be contained within 2% and that the bias for the target
value (veriable by participating in EQ programs with
switchable materials and titer assigned by the IFCC reference method) be minimal, with a total error target within 6%.
Postanalytical Aspects
As previously reported, the measurement of HbA1c is also
used to conrm the diagnosis of diabetes: values
≥48 mmol/mol (6.5%), if conrmed on at least two subsequent occasions, would allow the diagnosis of diabetes to
be made. HbA1c values between 43 and 47 mmol/mol
(between 6.1 and 6.4%) are associated with a high risk of
diabetes.
The DCCT study found that every 10mmol/mol (1%)
increase in HbA1c is associated with a worsening of mean
blood glucose of approximately 35 mg/dL, and current
ADA- recommended treatment targets indicate that the
primary goal of therapy should result in an HbA1c value
of no more than 53mmol/mol (7%). If HbA1c is consistently above 64 mmol/mol (8%), therapeutic treatment
should be promptly reassessed. Other organizations, such
as the IDF, recommend achieving and maintaining HbA1c
values below 48mmol/mol (6.5%) to minimize the risk
of complications. Another typical use of HbA1c is to measure the quality of care provided to patients with diabetes. The ADA criteria indicate that the percentage of
patients with HbA1c values greater than 75 mmol/mol
(9%) should not exceed 20% of the total adult patients;
the share of diabetics with values less than 53mmol/mol
(7%) should be at least 40% of the total; 84% of pediatric
patients should have HbA1c values less than 86mmol/mol
(10.0%) and 34% should have values less than 64mmol/
mol (8.0%).
Glycated Albumin andFructosamine
While fructosamine represents an index of the concentration
of stably glycated serum proteins (ketamine, mainly albumin, lipoproteins, and glycated globulins), GA represents a
unique post-translational product of albumin alone with
plasma glucose.
Although HbA1c remains the gold standard for the diagnosis and monitoring of diabetes mellitus, several studies have
shown that other biomarkers of nonenzymatic glycation,
such as GA and fructosamine, can be used both in clinical
conditions in which HbA1c dosage does not reect glycemic
compensation (chronic renal failure, anemia, and hemoglobin variants) and in other clinical settings in which information on blood glucose in the previous 2–3weeks is needed
(poorly controlled diabetes, gestational diabetes, postprandial hyperglycemia, anemia, hemoglobin variants).
Moreover, in other clinical settings, it is necessary to have
information on blood glucose in the previous 2–3 weeks
(poorly controlled diabetes, gestational diabetes, postprandial hyperglycemia, “uctuating” diabetes, and gastrectomized patients). GA and fructosamine are particularly
sensitive to recent changes in blood glucose and sudden
uctuations.
Preanalytical Aspects
Using these markers in clinical practice requires considering
some preanalytical variables that may affect the result. The
literature regarding this aspect is quite limited and sometimes conicting. In summary, all those clinical conditions
that alter the normal metabolism of albumin limit the use of
fructosamine and GA: thyroid dysfunction, nephrotic syndrome, liver cirrhosis, and nonalcoholic steatohepatitis
(NASH). Nephrotic syndrome, hyperthyroidism, NASH,
Cushing’s syndrome, and glucocorticoid intake decrease in
AG and fructosamine values, while liver cirrhosis and hypothyroidism determine an increase in the values of these
markers. Physiological or lifestyle variables such as age,
gender, race, body mass index (BMI), hyperuricemia, and
cigarette smoking also inuence the values of these
markers.
The intra- and interindividual biological variability of GA
was 2.1% and 10.6%, while that of fructosamine was 2.3%
and 6.3%.
GA values have an inverse correlation with BMI, fat mass
levels, and cigarette smoking, while they correlate positively
with age and are higher in females. Some authors also report
an increase in GA values throughout pregnancy, while others
report a decrease in values in the third trimester.
Fructosamine values show a modest decrease with gestational age and a modest increase with maternal age and correlate inversely with smoking. Higher values are found in
males, and differences have been reported in different races.
Regarding the biological matrix used for assaying these
markers, serum or plasma can be used. However, it has been
reported that the values found in plasma are lower than those
obtained in serum for fructosamine. It is therefore recommended that the same biological matrix is always used for
monitoring.
As shown in two studies, GA is stable for 24h on separated serum and plasma stored at room temperature, up to
1week when stored at 4°C, and up to 2months when stored
at −80°C.
Stability for up to 2weeks with serum stored at 4°C and
up to 5weeks when stored at −20°C has been demonstrated
for fructosamine.
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409
Analytical Aspects
Several methods have been developed to evaluate fructosamine in serum and plasma. The most widely used and best
standardized are colorimetric assays that typically exploit
the property of fructosamine to be a reducing agent under
alkaline conditions. The rst technique, developed in 1983,
was based on reducing the nitroblue tetrazolium dye to
formazan. The spectrophotometric method monitored the
rate of formazan formation, which was directly proportional
to the fructosamine concentration. Later, a nonionic detergent containing uricase was added to eliminate interference
from uric acid and polylysine, thus allowing a more accurate
and sensitive measurement. The assay thus modied is currently available and widely used in clinical laboratories.
Although it is rapid, technically simple, inexpensive, and
available for automation, the method is affected by ambient
temperature variations and remains poorly standardized. In
addition, many molecules with reducing activity, such as
bilirubin and vitamins, may interfere with the measurement.
GA concentration can be measured by boronate afnity
chromatography, ion exchange chromatography, HPLC, and
immunoassays (ELISA or radioimmunoassays). An enzymatic method with good analytical performanceand excellent specicity has recently been automated (Lucica GA-L
kit, Asahi Kasei Pharma, Tokyo, Japan). The method is based
on the initial elimination of endogenous glycated amino
acids and peroxides by a ketamine oxidase and a peroxidase.
The GA is then hydrolyzed by an albumin-specic proteinase, and the products of this reaction are oxidized by a ketamine oxidase. The H2O2 produced is then measured
quantitatively by a colorimetric method. In parallel, the concentration of albumin is measured by the bromocresol violet
method so that the nal result can be expressed as the ratio of
GA to total albumin. This method can be implemented on
major automated analytical platforms and offers good analytical performance.
insulin therapy, to verify the secretory capacity of β cells, it
is not possible to directly evaluate insulinemia because the
insulin administered as a drug would also be measured. In
these cases, it is indicated to evaluate the concentration of
C-peptide (which is not contained in the pharmacological
insulin) to verify the residual capacity of β-cells to produce
endogenous insulin.
Measurement of the C-peptide response to glucagon may
help in rare cases where it is difcult to differentiate the
diagnosis between type 1 and type 2 diabetes.
The determination of C-peptide is essential in evaluating
possible self-induced hypoglycemia due to nontherapeutic
insulin intake.
Analytical Aspects
Immunometric methods can measure insulin and C-peptide,
but despite the efforts of the scientic community, to date,
there are no standardized methods.
One of the analytical problems affecting these analytes’
measurements is cross-reactivity with insulin analogs.
Postanalytical Aspects
Insulin reference ranges depend on the method used and
must therefore be dened by each laboratory. After an overnight fast, the insulin concentration in a healthy, nonobese
subject is usually between 12 and 150pmol/L.ForC-peptide,
reference ranges have not been dened with certainty, and
different laboratories expressC- peptide values with different units (nmol/L, pmol/L, or ng/mL). The concentration of
C-peptide in the healthy population is usually between 0.78
and 1.89ng/mL (0.25–0.6 nnmol/L).
Recommended Readings
Insulin andC-Peptide
Routine testing of insulin and C-peptide is not recommended
in most patients with diabetes. However, the assay of these
analytes is indicated in the evaluation of fasting hypoglycemia to exclude the presence of an insulinoma. The diagnosis
of insulinoma is based on the persistence of an elevated
plasma insulin concentration associated with a decreased
glucose concentration. Insulin determination is also helpful
in evaluating and managing patients with polycystic ovary
syndrome.
C-Peptide is a fragment of the original molecule from
which insulin is formed. When endogenous insulin is produced, C-peptide is also released. In patients undergoing
American Diabetes Association (2016) Classication and diagnosis of
diabetes. Sec. 2. In Standards of Medical Care in Diabetes– 2016.
Diabetes Care 39(Suppl. 1):S13–S22
American Diabetes Association. 5 (2016) Glycemic targets. In
Standards of Medical Care in Diabetes-2016. Diabetes Care
39(Suppl. 1):S39–S46
Bonetti G, Carta M, Lapolla A, et al (2018) Raccomandazioni per
l’ottimizzazione della fase pre-analitica per una corretta determinazione della glicemia in ambito diabetologico. Biochimica Clinica
(in press) https://doi.org/10.19186/BC_2018.037
Carta M, Mosca A, Lapolla A et al (2015) Raccomandazioni per
l’esecuzione del test da carico orale di glucosio (OGTT). Biochimica
clinica 39:135–140
Graziani MS, Caldini AL (2011) per il GdS intersocietario
Diabete. Indicazioni per la misura dell’albumina nelle urine per
l’accertamento e il monitoraggio della nefropatia diabetica. Riv Ital
Med Lab 7:20–24
410
https://t.me/medicina_free
A. Mosca and M. Montagnana
http://www.sibioc.it/gds/more/sezione- id/7/tag/Documenti%20
Ufciali
International Diabetes Federation IDF guidelines for management of
postmeal glucose in patients with diabetes. [Accessed 6 Dec 2016].
Available from: http://www.idf.org/sites/default/les/postmeal%20
glucose%20guidelines.pdf
Lapolla A, Mosca A (2012) Screening e diagnosi del diabete gestazio-
nale: denite le raccomandazioni. Biochimica Clinica 36:12–14
Mosca A, Lapolla A (2008) Diagnostica del diabete mellito. In:
Interpretazione degli esami di laboratorio, a cura di Mauro
Panteghini. Trattato italiano di Medicina di Laboratorio, vol
VII.Padova: Piccin Nuova Libraria, pp85–133
Sacks DB, Arnold M, Bakris GL etal (2011) National Academy of
Clinical Biochemistry; Evidence-Based Laboratory Medicine
Committee of the American Association for Clinical Chemistry.
Guidelines and recommendations for laboratory analysis in the
diagnosis and management of diabetes mellitus. Diabetes Care
34:e61–e99
Sacks DB, Bruns DE, Goldstein DE etal (2002) Guidelines and recom-
mendations for laboratory analysis in the diagnosis and management of diabetes mellitus. Clin Chem 48:436–472
Standard italiani per la cura del diabete mellito (2018). www.standar-
ditaliani.it
Metabolic Syndrome
https://t.me/medicina_free
MarcelloCiaccio, AnnaMariaCiaccio, andLuisaAgnello
28
Introduction
Metabolic syndrome is a biochemical–clinical condition
characterized by resistance to insulin action associated with
a proinammatory and prothrombotic state. It represents an
important cardiovascular risk factor with a constantly
increasing incidencein the general population; therefore, its
early detection is fundamentalto implement preventive interventions. This chapter describes the main characteristics of
metabolic syndromeand thecriteria used to diagnose it.
Denition
Metabolic syndrome is a condition in which the interaction
among biochemical, clinical, and metabolic factors results in
an increased risk of developing cardiovascular diseases, type
2 diabetes mellitus, and mortality. It is present in about
20–25% of the general population and is characterized by
visceral obesity, hypertension, and lipid and glucose metabolism alterations.
M. Ciaccio (*)
Department of Biomedicine, Neurosciences and Advanced
Diagnostics, Institute of Clinical Biochemistry, Clinical Molecular
Medicine and Clinical Laboratory Medicine, University Hospital
“P.Giaccone”, Palermo, Italy
Department of Laboratory Medicine, University Hospital
“P.Giaccone”, Palermo, Italy
e-mail: marcello.ciaccio@unipa.it
A. M. Ciaccio
Department of Health Promotion, Mother and Child Care, Internal
Medicine and Medical Specialties (ProMISE) “G.D’Alessandro”,
University Hospital “P.Giaccone”, Palermo, Italy
L. Agnello
Department of Biomedicine, Neurosciences and Advanced
Diagnostics, Institute of Clinical Biochemistry, Clinical Molecular
Medicine and Clinical Laboratory Medicine, University Hospital
“P.Giaccone”, Palermo, Italy
Beginning with its initial description as the “android
obesity syndrome” by Vague in 1956, the syndrome has
taken on several names over time, including “dyslipidemic hypertension syndrome,” “syndrome X,” “insulin
resistance syndrome,” “dysmetabolic syndrome,” and
“plurimetabolic syndrome.”
Pathogenesis
The metabolic syndrome has a multifactorial etiology that
includes genetic and environmental factors. The underlyingpathophysiological mechanismis insulin resistance (IR),
dened as an inadequate response of target organs (striated
muscle, liver, and adipose tissue) to the physiological effects
of insulin.
The consequences of reduced insulin sensitivity are:
1. Reduced insulin-mediated uptake of glucose in striated
muscle and adipose tissue.
2. Reduced insulin-mediated inhibition of glucose produc-
tion by the liver.
3. Signicant reduction in lipolysis inhibition in adipose
tissue.
Obesity, physical inactivity, and genetic factors predispose to the development of IR.In particular, the most important cause of IR is the increase of adipose tissue, especially
at the abdominal-perivisceral level. Obesity alters mechanisms that regulate the activity of the insulin receptor.
Specically, mediators, such as the excess of circulating
freefatty acids (FFAs) andtumor necrosis factor-alpha“shutdown” the receptor’s activity.
Abdominal-visceral obesity results in an overow of FFA
into the circulation and a consequent increase in FFA uptake
© The Author(s), under exclusive license to Springer Nature Switzerland AG 2023
M. Ciaccio (ed.), Clinical and Laboratory Medicine Textbook, https://doi.org/10.1007/978-3-031-24958-7_28
411
412
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by muscle tissue, liver, and adipose tissue. FFAs are normally metabolized through two pathways:
• Oxidation
• Deposition (triglycerides)
However, when the overow of FFAs exceeds the metabolic capacity of these two pathways, FFAs and their metabolites accumulate in the tissues, where they cause the
activation of specic serine kinases, which, in turn, act by
blocking the insulin signal.
Diagnosis
Currently, there are no single criteria for diagnosing metabolic syndrome, but scientic societies and organizations
have proposed several. In general, the diagnosis of metabolic
syndrome is based on the evaluation of biochemical parameters, such as glycemia and lipid alterations, and clinical
parameters, such as arterial hypertension and obesity. The
different diagnostic criteria proposed so far for metabolic
syndrome are shown in Table28.1.
Although IR represents the pathogenic mechanism underlying the metabolic syndrome, the assessment of this conditionis not recommended for diagnosis. However, once the
diagnosis of metabolic syndrome is made, several tests are
available to assess the insulin resistanceseverity.
The fasting insulin measurement is not reliable because
insulin may have normal values leading to false negative
results. IR can be assessed by direct or indirect methods. The
direct methods are the insulin measurement during the oral
glucose tolerance test (OGTT) curve, the insulin suppression
test, and the hyperinsulinemic-euglycemic clamp, which is
the gold standard. Among the indirect methods, two indices
are commonly used: the Homeostatic Model Assessment
(HOMA) index and the Quantitative Insulin sensitivity
Check Index (QUICKI). OGTT is performed after fasting for
8–10h (overnight fasting) by giving the patient 75g of glucose dissolved in 300mL of water. Insulin measurement is
performed before the administration of the glucose solution
(basal) and thenat precise time intervals (30′, 60′, 120′, 150′,
and 180′). In healthy subjects, insulinemia reaches a concentration peak 6–10 times higher than the basal value after
30–60min, then its levels begin to decrease, reaching a value
approximately 2–3 times the basal value around 180min and
normal around 240min (Fig.28.1). In a subject with insulin
resistance, the peak at 30–60min will reach higher concentrations than in a subject and persist for a longer period
because insulin is metabolized more slowly (Fig.28.1).
The insulin suppression test is based on administering an
octreotide and somatostatin solution to suppress insulin
Table 28.1 Diagnostic criteria of the metabolic syndrome.
AHA-NHLBI IDF NCEP-ATPIII WHO
Criterion Any combination of three
Glycemic changes FPG >100mg/dL or overt
Insulin resistance M-clamp of the lower quartile of
Central obesity Waist circumference
Hypertension
Hypertriglyceridemia
Low C-HDL <40mg/dL in men and
Dyslipidemia
Microalbuminuria
AHA, American Heart Association; BMI, body mass index; HDL-C, high-density lipoprotein cholesterol; FPG, fasting plasma glucose; IDF,
International Diabetes Federation; IGT, impaired glucose tolerance; NCEP-ATP III, National Cholesterol Education Program Adult Treatment
Panel III; NHLBI, National Heart, Lung, and Blood Institute; WHO, World Health Organization.
altered parameters
diabetes
≥102cm in men and
≥88cm in women
Systolic ≥130 and/or
diastolic ≥85mmHg or
antihypertensive treatment
≥150mg/dL or lipidlowering treatment
<50mg/dL in women, or
lipid-lowering treatment
Central obesity and two
other altered parameters
FPG >100mg/dL or overt
diabetes
Waist circumference
≥94cm in men and
≥80cm in women
Systolic ≥130 and/or
diastolic ≥85mmHg or
antihypertensive treatment
≥150mg/dL or lipidlowering treatment
<40mg/dL in men and
<50mg/dL in women, or
lipid-lowering treatment
Any combination of three
altered parameters
FPG >100mg/dL or overt
diabetes
Waist circumference
≥102cm in men and
≥88cm in women
Systolic ≥130 and/or
diastolic ≥85mmHg
≥150mg/dL
<40mg/dL in men and
<50mg/dL in women, or
lipid-lowering treatment
Glycemic alterations or insulin
resistance and two further altered
parameters
FPG>100mg/dL or IGT or overt
diabetes
the distribution
Waist/hip ratio
>0.90in men and
>0.85in women and/or BMI ≥30
Systolic ≥140 and/or
diastolic ≥90mmHg
Triglycerides ≥150mg/dL and/
or HDL-C<35mg/dL in men
and HDL-C<39mg/dL in
women
Microalbuminuria (>20μg/min)
or albuminuria/creatinuria
ratio>30μg/mg
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