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Monitoring of Patients with Diabetes




Guidelines & Recommendations for Laboratory Analysis in the Diagnosis & Management of Diabetes Mellitus


David Goldstein, MD
University of Missouri
Columbia, MO

I.                     USE

[Recommendations:  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.]

Measurement 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).

The American 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).

II.                   RATIONALE

Glycated proteins 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 established (2,3). 

III.                  ANALYTICAL

A.     Background

[Recommendations:  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 website:

Pre-analytical Issues

1.      Patient Variables

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 interference.

Some vitamins 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 method.


2.      Sample 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.

Recently, a 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.

B.     Analytical Issues

1.        Performance goals and quality control

Several expert 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.

The laboratory 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% be investigated.

2.        Reference interval

The laboratory 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.

3.        Out-of-range specimens

The laboratory 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.

4.        Linearity testing

The laboratory should perform linearity testing initially and annually thereafter.

5.        Removal of labile GHB

Formation of 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 removal.

IV.               INTERPRETATION

A.                 Laboratory-physician interface

The laboratory 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.

B.                 Clinical application

Treatment goals: 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).

Testing frequency: 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).

Interpretation:  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.


A.                 Use of GHB for diabetes screening/diagnosis

At present, 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%).

B.                 Use of other GPs including advanced glycation end-products for routine management of diabetes mellitus

Further studies 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).

C.                Global 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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