& Recommendations for Laboratory Analysis in the Diagnosis & Management
of Diabetes Mellitus
University of Missouri
Glycated hemoglobin (GHB) should be measured routinely in all patients
with diabetes mellitus to document their degree of glycemic control.
Treatment goals should be based on the results of prospective randomized
clinical trials such as the Diabetes Control and Complications Trial
and the United Kingdom Prospective Diabetes Study which have documented
the relationship between glycemic control, as quantified by serial
determinations of GHB, and risks for the development and progression
of chronic diabetes complications.]
of glycated proteins (GP), primarily glycated hemoglobin (GHB),
is widely used for routine monitoring of long-term glycemic status
in patients with diabetes mellitus. The test is used both as an
index of mean glycemia and as a measure of risk for the development
of diabetes complications (1-3). This test is also being used increasingly
by quality assurance programs to assess the quality of diabetes
care (e.g., requiring that health-care providers document the frequency
of GHB testing in patients with diabetes, the proportion of patients
with GHB values below a specified level)(4,5).
Diabetes Association (ADA) recommends measurement of GHB in patients
with both type 1 and type 2 diabetes, first to document the degree
of glycemic control, then as part of continuing care (1). The ADA
has recommended specific treatment goals for GHB based on the results
of prospective randomized clinical trials, most notably the Diabetes
Control and Complications Trial (DCCT)(6,7), but also the more recently
completed United Kingdom Prospective Diabetes Study (UKPDS)(8,9).
Since different GHB assays can give varying GHB values, the ADA
recommends that laboratories use only assay methods that are certified
as traceable to the DCCT GHB reference (1,2).
are formed post-translationally from the slow, non-enzymatic reaction
between glucose and amino groups on proteins (10). For hemoglobin,
the rate of synthesis of GHB is principally a function of the concentration
of glucose to which the erythrocytes are exposed. Studies have
demonstrated that GHB is a clinically useful index of mean glycemia
during the preceding 120 days, the average lifespan of erythrocytes
(3,10-17). Although carefully controlled studies have documented
a close relationship between the level of GHB and mean glycemia,
routine determinations of blood glucose by patients or by their
health-care providers are not considered as reliable as GHB to quantify
mean glycemia (3,11,12,18-21). Levels of other blood-based glycated
proteins (e.g. glycated serum/plasma proteins, “fructosamine”) also
reflect mean glycemia, but over a much shorter time than GHB (15-30
days and 60-120 days, respectively)(3,10-18,22,23). Clinical utility
of glycated proteins other than hemoglobin has not been clearly
Laboratories should use only GHB assay methods that are certified
by the National Glycohemoglobin Standardization Program as traceable
to the DCCT reference. In addition, laboratories that measure GHB
should participate in a proficiency testing program, such as the
College of American Pathologists Glycohemoglobin Survey, that uses
fresh blood samples with targets set by the National Glycohemoglobin
Standardization Program Laboratory Network.]
There are many
(greater than 30) different GHB assay methods in current use. These
range from low throughput research laboratory component systems
and manual minicolumn methods to high throughput automated systems
dedicated to GHB determinations. Most methods can be classified
into one of two groups based on assay principle (3,13,24). The
first group includes methods that quantify GHB based on charge differences
between glycated and nonglycated components. Examples include cation-exchange
chromatography and agar gel electrophoresis. The second group includes
methods that separate components based on structural differences
between glycated and nonglycated components. Examples include boronate
affinity chromatography and immunoassay. Most charge-based and
immunoassay methods quantify hemoglobin A1c, defined as hemoglobin
A with glucose attached to the NH2-terminus valine of one or both
beta chains. Other methods quantify “total glycated hemoglobin,”
which includes both hemoglobin A1c and other hemoglobin-glucose
adducts (e.g., glucose-lysine adducts and glucose-alpha chain NH2-terminus
valine adducts). Generally, results between methods using different
assay principles show excellent intercorrelation and there are no
convincing data to show that any one method or analyte is clinically
superior to any other. However, the reported GHB results from the
same blood sample could differ considerably among methods unless
they are standardized to a common reference (e.g., without standardization,
the same blood sample could be read as 7% in one laboratory and
9% in another) (3,13,24-30).
In 1996, the
National Glycohemoglobin Standardization Program (NGSP) was initiated
to standardize GHB test results among laboratories to DCCT-equivalent
values (29-31). The rationale for standardizing GHB test results
to DCCT values was that the DCCT had determined the relationship
between specific GHB values and long-term outcome risks in patients
with diabetes mellitus(1-3,6). The NGSP was developed under the
auspices of the American Association for Clinical Chemistry and
is endorsed by the American Diabetes Association (ADA), which recommends
that laboratories use only GHB methods that have passed certification
testing by the NGSP. In addition, the ADA recommends that all laboratories
performing GHB testing participate in the College of American Pathologists
proficiency testing survey for GHB started in mid-1996, which uses
fresh whole-blood specimens (32-34).
The NGSP laboratory
network includes a variety of assay methods, each calibrated to
the DCCT reference. The DCCT reference is a high-performance liquid
chromatographic cation-exchange method that quantifies hemoglobin
A1c and is a NCCLS designated comparison method(35,36). The assay
method has been on-line since 1978 and has demonstrated long-term
high precision (between-run CVs consistently <3%). The laboratories
in the network interact with manufacturers of GHB methods to assist
them, first in calibrating their methods, and then in providing
comparison data for certification of traceability to the DCCT.
The certification process involves evaluation of both precision
and accuracy using specific criteria. Certification is valid for
one year. An important adjunct to the program is the GHB proficiency
testing survey administered by the College of American Pathologists
(CAP) in which more than 2000 laboratories participate. Since 1996
(starting with a pilot project including 500 laboratories and expanded
to all laboratories in 1998), the survey has utilized fresh blood
samples with NGSP-assigned target values. Since initiation of the
NGSP in 1996, the survey has documented a steady improvement in
comparability of GHB values among laboratories, both within-method
and between-method. In general, NGSP-certified methods have demonstrated
less variability (based on between-laboratory CVs) and better comparability
to NGSP-assigned target values than non-certified methods (32-34).
The NGSP website provides detailed information on the certification
process and maintains a listing of certified assay methods (NGSP
There are no
clinically significant effects of age, sex, ethnicity, or season
on GHB test results. Results are also not significantly affected
by acute illness. Any condition (e.g., hemolytic anemia, blood
loss) that shortens erythrocyte survival will falsely lower test
results (3). There is no significant effect of food intake on test
results. Hypertriglyceridemia, hyperbilirubinemia, and uremia can
interfere with some assay methods (13). A number of hemoglobinopathies
(e.g., hemoglobins S, C, Graz, Sherwood Forest, D, Padova) are also
reported to interfere with some assay methods (37,38, 39). Some
methods may give a value in the normal range for a nondiabetic patient
with a hemoglobin variant, but this is not an assurance that no
interference is present; the interference may be subtle in the normal
range but increase steadily with increasing GHB. Boronate affinity
chromatographic assay methods are generally considered to be less
affected by hemoglobinopathies than methods that separate glycated
and nonglycated components based on charge differences. In some
instances, such as with most cation-exchange high performance liquid
chromatographic methods, manual inspection of chromatograms can
alert the laboratory to the presence of either a variant or a possible
and medications are reported to interfere with test results with
some assay methods. These include vitamins C and E, and salicylates
(40-42). Test results may also be affected in individuals with
alcoholism and opiate addiction (43,44).
Since interferences are method specific, product instructions
from the manufacturer should be reviewed before use of the GHB assay
collection, handling, and storage
Blood can be
obtained by venipuncture or by fingerprick capillary sampling (45,46).
Blood tubes should contain anticoagulant as specified by the manufacturer
of the GHB assay method (EDTA can be used unless otherwise specified
by the manufacturer). Sample stability is assay method specific
(47,48). In general, whole blood samples are stable for up to 1
week at 4 degrees C. For most methods, whole blood samples stored
at –70 degrees C or colder are stable long-term (at least one year)
but with most assay methods, specimens are not stable at –20 degrees
C. Improper handling of specimens such as storage at high temperatures,
can introduce large artifacts that may not be detectable, depending
on the assay method.
number of convenient blood collection systems have been introduced,
including filter paper and small vials containing stabilizing/lysing
reagent (49-51). These systems are designed for field collection
of specimens with routine mailing to the laboratory. These systems
are generally matched to specific assay methods and should be used
only if studies have been performed to establish comparability of
test results using these collection systems with standard sample
collection and handling methods for the specific assay method employed.
goals and quality control
groups have presented recommendations for assay performance. Early
reports recommended that interassay coefficients of variation (CV)
be < 5% at normal and diabetic GHB levels (52). More recent
reports suggest lower CVs (e.g., intralaboratory <3% and interlaboratory
<5% (53). These recommendations are reasonable; many current
assay methods were addressed CVs <3%. Regardless of the assay
method, the laboratory should strive to achieve as low a CV as possible
to optimize clinical utility of the test.
should include 2 levels (high and low) of controls at the beginning
and end of each day’s run. Frozen whole blood controls stored at
–70 degrees C or colder in single use aliquots are ideal and are
stable for months or even years depending on the assay method.
Lyophilized controls are commercially available, but depending on
the assay method, may show matrix effects when new reagents or columns
are introduced. It is recommended that the laboratory consider
using both commercial and in-house controls to optimize performance
monitoring. The laboratory should establish strict criteria for
acceptance or rejection of assay runs with limits of acceptability
for individual runs and for long-term trends. (54,55,56)
It is also recommended
that the laboratory analyze split-duplicate samples (2-5% of samples)
routinely in addition to controls as an independent measure of assay
performance. Criteria for acceptability of duplicate results will
depend on the inherent assay CV. For assays with CVs <3%, it
is recommended that split-duplicate specimens that differ by >5%
should determine its own reference interval according to NCCLS guidelines
(NCCLS Document C28A) even if the manufacturer has provided one.
Test subjects should be nonobese and have fasting plasma glucose
<110 mg/dL. For NGSP-certified assay methods, the SD for the
reference interval is generally 0.5% GHB or less, resulting in a
95 % confidence interval (mean +/- 2 SD) of 2 % GHB or lower (e.g.,
mean hemoglobin A1c +/- 2 SD = 5.0 +/- 1.0%). For NGSP-certified
methods, an individual laboratory-determined reference interval
outside this range should be investigated.
should repeat testing for all sample results below the reference
range or above the range of documented linearity. In addition,
sample results greater than 14% GHB should be repeated.
The laboratory should perform linearity testing initially and
of labile GHB
GHB includes an intermediate Schiff-base which is called “pre-A1c”
or labile (57,58). This material is formed rapidly with hyperglycemia
and interferes with some GHB assay methods, primarily those that
are charge-based. For methods that are affected by this labile
intermediate, manufacturer instructions should be followed for its
should work closely with physicians who order GHB testing. Proper
interpretation of test results requires an understanding of the
assay method, including its known interferences. For example, if
the assay method is affected by hemoglobinopathies (independent
of any shortened erythrocyte survival) or uremia, the physician
should be made aware of this.
If the assay
method is NGSP-certified, the physician should be provided with
information (preferably on the test report) relating GHB test results
to mean glycemia and outcome risks as determined by the DCCT (2,3,6,14).
This information is available on the NGSP website.
There is recent
interest by physicians and their patients with diabetes to have
GHB test results available at the time of the clinic visit. Studies
suggest that immediate feedback to patients with GHB test results
improves their long-term glycemic control (59). Further studies
are needed to confirm these findings. It is possible to achieve
the goal of having GHB test results available at the time of the
clinic visit by either having the patient send in a blood sample
1-2 weeks before the scheduled clinic visit or by having a rapid
throughput assay system convenient to the clinic.
GHB measurements are now a routine component of the clinical management
of patients with diabetes mellitus. Based principally on the results
of the DCCT, the ADA has recommended that a primary goal of therapy
is a GHB level < 7%, and that physicians should reevaluate the
treatment regimen in patients with GHB values consistently >8%
(1,2). These GHB values apply only to assay methods that are certified
as traceable to the DCCT reference, with reference interval approximately
4-6% hemoglobin A1c or hemoglobin A1c-equivalent. In the DCCT,
each 10% lowering of GHB (e.g., 12 vs. 10.8% or 8 vs. 7.2%) was
associated with approximately 45% lower risk for the progression
of diabetic retinopathy (60). Similar risk reductions were found
in the UKPDS (7,8).
There is no consensus on the optimal frequency of GHB testing.
The ADA recommends (2): “that for any individual patient, the frequency
of GHB testing should be dependent on the judgment of the physician.
In the absence of well-controlled studies that suggest a definite
testing protocol, expert opinion recommends GHB testing at least
two times a year in patients who are meeting treatment goals (and
who have stable glycemic control) and more frequently (quarterly
assessment) in patients whose therapy has changed or who are not
meeting glycemic goals.” Diabetes quality assurance programs (e.g.,
ADA Provider Recognition Program and HEDIS 2000 (4,5) have generally
required documentation of the percent of patients with diabetes
who have had at least one GHB determination during the past year.
Studies have established that serial (quarterly for one year) measurements
of GHB result in large improvements in GHB values in patients with
type 1 diabetes (61).
GHB values in patients with diabetes are a continuum; they range
from normal in a small percentage of patients whose mean blood glucose
levels are in or close to the normal range, to markedly elevated
values, e.g., two-to-threefold increases, in some patients, reflecting
an extreme degree of hyperglycemia. Proper interpretation of GHB
test results requires that physicians understand the relationship
between test results and mean blood glucose, the kinetics of GHB,
and specific assay limitations/interferences (3). Small changes
in GHB (e.g., +/- 0.5% GHB) over time may reflect assay
variability rather than any true change in glycemic status.
of GHB for diabetes screening/diagnosis
the ADA does not recommend GHB for diabetes screening or diagnosis
(62). There is considerable controversy regarding this issue and
further studies are needed to determine if GHB is useful for screening
and/or diagnosis of diabetes (62-66). Certainly, standardization
of GHB ssays has obviated one of the most commonly stated reasons
for not using GHB for screening and/or diagnosis. Optimal clinical
utility of GHB for screening and/or diagnosis will also require
highly precise assay methods (e.g., intralaboratory CVs < 3%).
Use of other GPs including advanced glycation
end-products for routine management of diabetes mellitus
are needed to determine if other GPs such as fructosamine are clinically
useful for routine monitoring of patients’ glycemic status. Further
studies are also needed to determine if measurements of advanced
glycation end-products (AGEs) are clinically useful as predictors
of risk for chronic diabetes complications (67).
standardization of GHB testing
In 1995, the
International Federation of Clinical Chemistry (IFCC) formed a Working
Group on HbA1c Standardization (IFCC-WG). This committee, which
includes members from the NGSP Steering Committee and Laboratory
Network, has been evaluating several candidate reference methods
and purified GHB materials (purified HbA1c) that potentially could
provide firm links between the NGSP and GHB standardization programs
in other countries (68). Such a scheme is particularly attractive
since it would allow GHB test results world-wide to be comparable
to those in the DCCT and the more recently completed UKPDS. The
IFCC has established a laboratory network using both mass spectroscopy
and capillary electrophoresis as candidate reference methods. The
candidate reference material is a mixture of highly-purified HbA1c
and HbAO (69-71). Initial comparisons between samples analyzed
by the IFCC Laboratory Network and the NGSP Laboratory Network are
encouraging; there appears to be a linear relationship between the
two reference systems (personal communication with Kornelius Miedema,
Chairholder IFCC-WG, 17 January 2000). If further studies confirm
a consistent relationship between the two networks, it will be possible
to use one of the IFCC reference methods to replace the current
NGSP anchor (a designated comparison method with far less specificity
for HbA1c than either the mass spectroscopy or capillary electrophoresis
methods). Assuming that the IFCC reference system is adopted by
the NGSP and other standardization programs, an important issue
that would need to be addressed is that of the GHB numbers. The
IFCC reference system GHB numbers are on a different scale than
the DCCT GHB numbers. Should the new number system be adopted along
with the new reference system or should the current number scheme,
which is widely used, be retained (the IFCC GHB numbers could be
converted into DCCT numbers by an equation). Proper resolution
of this important question will require international consensus
with a process that includes both clinicians and laboratorians.
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