In 1979 a work group of the National Diabetes Data Group1
established the criteria, later endorsed by the World Health Organization
Committee on Diabetes, that patients with a fasting or 2-h postprandial
glucose concentration greater than 140 or 200 mg/dL, respectively, were
to be considered diabetic.
In 1997, the Expert Committee on the Diagnosis and Classification of
Diabetes Mellitus was convened to reexamine the classification and diagnostic
criteria for diabetes based on the 1979 publication of the National Diabetes
Data Group. As a result of its deliberations, the committee recommended
several changes to the diagnostic criteria for diabetes and for lesser
degrees of impaired glucose regulation.2
The use of a fasting plasma glucose, or FPG, test for the diagnosis of
diabetes was recommended, and the cut point separating diabetes from nondiabetes
was lowered from a FPG > 140 mg/dL to > 126 mg/dL.
This change was based on data that showed an increase in prevalence and
incidence of diabetic retinopathy beginning approximately at a FPG of
126 mg/dL, as well as on the desire to reduce the discrepancy that existed
in the number of cases detected by the FPG cut point of > 140
mg/dL and the 2-h value in the OGTT (2-h plasma glucose) of >
In November 2003, the Expert Committee on the Diagnosis and Classification
of Diabetes Mellitus3 recommended that
the criteria to diagnose diabetes remain as previously defined. However,
the lower cut point defining impaired fasting glucose, or IFG, was reduced
from > 110 mg/dL to > 100 mg/dL. Thus, "normal" would
now be defined as a FPG of < 100 mg/dL. These diagnostic criteria for
diabetes mellitus were restated in January 2005.4
Establishing a single number for glucose to determine whether a patient’s
value is "normal" or "abnormal"—that is, a fasting plasma glucose
of 100 mg/dL—is a way to help physicians remember when a patient’s
fasting glucose is increased; however, it does not take into account the
variability of the measurement itself.
There is a marked variability involved (both biologic and analytic)
in glucose testing. Physicians are taught in medical school that this
variability exists and that the biological variability is substantially
greater than the analytic variability.5
The concentration of glucose in the serum of a fasting individual is
not the same when measured on different days. The 2002 diabetes mellitus
practice guideline of the National Academy of Clinical Biochemistry5
cites Ollerton, et al’s study6 that reports
biological variabilities around 6.9 percent (coefficient of variation).
Thus, even using a theoretically perfect measurement method for glucose,
a patient with an average fasting glucose concentration of 126 mg/dL would
have actual (reported) concentrations spanning from 109 to 143 mg/dL in
19 out of 20 different days, and even higher than 143 and lower than 109
mg/dL in the remaining one out of 20 days. This variability would be worse
if the sample was obtained at different times of the day, since fasting
plasma glucose is higher in the morning than in the afternoon.7
Even greater variability in the measurement of glucose is introduced
by the manner in which the blood sample is collected and handled before
its arrival in the laboratory. While plasma is recommended as the preferred
specimen,4 some clinicians might be using
results obtained on serum specimens (the liquid portion of blood after
clotting). Serum intrinsically has glucose concentrations that are five
percent higher than plasma.8
Furthermore, while glucose is stable for 72 hours in refrigerated serum
or plasma that has been separated from blood cells by centrifugation,
it changes rapidly if the cells are allowed to remain in contact with
serum or plasma because the blood cells will continue to consume it. The
rate of disappearance of glucose in the presence of blood cells has been
reported to average 10 mg/dL per hour,9
but the rate increases with glucose concentration, temperature, white
blood cell count, and other factors.10
This problem can be attenuated by the addition of fluoride to the blood
collection tube (gray-top tube), but fluoride takes about one hour to
effectively stop the consumption of glucose by blood cells.11,12
The rates of decline of glucose in the first hour after sample collection
in tubes with and without fluoride are virtually identical.9
Since physician offices might separate plasma from cells at various
times after collection or submit the uncentrifugated (whole blood) original
fluoride tube for analysis, even a perfect test would underestimate to
a variable extent the amount of glucose in the sample.
Some of the analytic variables involved in the testing of glucose include
differences within the same lot of collection tubes, lot-to-lot variation
in collection tubes, lot-to-lot variation of reagents, calibration, and
Due to the variability of measuring glucose, in order to make a diagnosis
of diabetes, the American Diabetes Association, or ADA, recommended that
a patient’s glucose value be confirmed on a subsequent day to make certain
that the patient’s glucose value exceeded 126 mg/dL on more than one occasion.3,4
Instrument manufacturers establish their calibrator values for the glucose
assay using the National Institute of Standards and Technology material,
or material that has been standardized to NIST. Because of issues related
to the lack of availability of the NIST standard in 2003 and in part of
2004, manufacturers apparently assigned glucose calibrator values that
caused slightly elevated glucose results on patient samples. During this
period of time, laboratories were well within the total allowable error
as determined by both the CAP and the Clinical Laboratory Improvement
Amendments of 1988. CLIA allows a total allowable error of ± 6 mg/dL or
10 percent for glucose, whichever value is greater. We believe the minimal
positive shift caused by the slightly biased calibrator value assignment
had little or no effect on patient results, especially when considered
in the context of the significantly larger biological and preanalytical
variability affecting plasma glucose measurements, particularly the negative
bias due to the glycolytic activity of blood cells in the sample.
A review of results of CAP proficiency testing for glucose during this
period revealed that all laboratories using automated glucose platforms/systems
gave comparable results.
During this period, some physicians contacted laboratories saying they
had a higher number of patients with abnormally high glucose values. We
believe this was largely due to the stricter ADA classification for impaired
fasting glucose imposed in 2004 and not due to any analytical issues.
Currently, the National Cholesterol Education Program and the Centers
for Disease Control and Prevention jointly define the accuracy of laboratory
tests for lipids and the proper protocol for sampling. As a result, there
are standards against which laboratories are able to compare their lipid
test results. There is no similar oversight committee for glucose assays,
however, that defines appropriate preanalytical, analytical, and postanalytical
requirements for testing.
We need to ensure at a minimum that NIST standard material is plentiful
and is kept current. Physician awareness needs to be raised about the
numerous variables affecting glucose measurements. Some clinicians should
be reminded not to make the diagnosis of diabetes or impaired glucose
tolerance based on a single measurement and to consider laboratory results
in the clinical context.
We will need to work with the American Diabetes Association to assist
in recommending guidelines for the most optimal glucose samples and encourage
both the CAP and the ADA to consider forming an expert committee with
representatives from laboratories, manufacturers, and practicing physicians
to establish improved guidelines for glucose sample collection and processing.
This expert committee will need to develop a national system to monitor
and ensure the accuracy of glucose measurements and to address how to
appropriately interpret borderline results and followup actions.
Dr. Schwartz is vice president and chief laboratory officer; Dr. Reichberg
is corporate pathologist, science and technology; and Dr. Gambino is chief
medical officer emeritus—all at Quest Diagnostics, Corporate Medical
Department, Lyndhurst, NJ.
- National Diabetes Data Group. Classification and diagnosis of diabetes
mellitus and other categories of glucose intolerance. Diabetes.
- Expert Committee on the Diagnosis and Classification of Diabetes Mellitus.
Report of the expert committee on the diagnosis and classification of
Diabetes Care. 1997;20:1183-1197.
- Expert Committee on the Diagnosis and Classification of Diabetes Mellitus.
Follow-up report on the diagnosis of diabetes mellitus. Diabetes
- American Diabetes Association. Standards of medical care in diabetes.
- Sacks DB, Bruns DE, Goldstein DE, Maclaren NK, McDonald JM, Parrott
M. Guidelines and recommendations for laboratory analysis in the diagnosis
and management of diabetes mellitus. Clin
- Ollerton RL, Playle R, Ahmed K, Dunstan FD, Luzio SD, Owens DR. Day-to-day
variability of fasting plasma glucose in newly diagnosed type 2 diabetic
- Troisi RJ, Cowie CC, Harris MI. Diurnal variation in fasting plasma
glucose: Implications for diagnosis of diabetes in patients examined
in the afternoon. JAMA.
- Ladenson JH, Tsai LM, Michael JM, Kessler G, Joist JH. Serum versus
heparinized plasma for eighteen common chemistry tests: Is serum the
appropriate specimen? Am
J Clin Pathol. 1974;62:545-552.
- Chan AY, Swaminathan R, Cockram CS. Effectiveness of sodium fluoride
as a preservative of glucose in blood. Clin
- Ladenson JH. Nonanalytical sources of variation in clinical chemistry
results. In: Sonnenwirth A, Jarett L, eds. Clinical Laboratory Methods
and Diagnosis. St. Louis, Mo.: CV Mosby; 1980:149-192.
- Le Roux CW, Wilkinson SD, Pavitt DV, Muller BR, Alaghband-Zadeh J.
A new antiglycolytic agent. Ann
Clin Biochem. 2004;41(Pt1):43-46.
- Foucher B, Pina G, Desjeux G, Cheminel V, Prevosto JM. Stability of
glucose in blood collected with or without antiglycolytic agent. Ann
Biol Clin (Paris). 2004;62(5):601-604.