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Diabetic Testing and Monitoring

Reprint from Focus On, 1998, Issue 6

Throughout the history of diabetes, laboratory test values have played an important role in the diagnosis and treatment of the disease. In this article we will explore the historic use of the laboratory testing, criteria for diagnosing diabetes mellitus (DM), and recent testing developments yet to find their clinical niche.

Prior to 1997, diagnostic tests for DM usually included the use of an Oral Glucose Tolerance Test (OGTT) performed in accordance with World Health Organization Guidelines. This test was difficult for both the patient and technician performing the test. Prior to testing, the patient was on an unrestricted diet of greater than 150g of carbohydrate for three successive days. The patient was also expected to have unlimited physical activity for the 3 day period. The day prior to the test, the patient was required to fast for 10 to 16 hours with minimal water permitted. Once at the laboratory, a fasting blood glucose would be collected. The patient would then be administered an oral glucose beverage containing 75 grams of glucose for a non pregnant adult, 1.75g of glucose per kilogram of ideal body weight for children, or 100g for pregnant women. The drink was to be consumed within five minutes and the timed testing would begin. Blood was collected for glucose levels every 30 minutes for two hours (the customary end point for the test). The patients were expected to remain seated for the entire testing period. This was difficult for pediatric or geriatric patients to maintain.

A negative diagnosis for DM was definitely confirmed with a negative Oral Glucose Tolerance Test. However, there are numerous factors which can affect the test and induce a false positive result. Any source of stress, physical, psychosocial, or pathophysiologic factors could contribute to a false positive result. The 1997 American Diabetes Association (ADA) guidelines, emphasized utilizing the fasting plasma glucose instead of the oral glucose tolerance test to diagnose DM.

According to the 1997 ADA guidelines, at least one of the following criteria must be met to qualify for the diagnosis of DM:

  • Random serum or plasma glucose $ 200 mg/dL and physical symptoms
  • Eight hour fasting serum or plasma glucose $ 126 mg/dL
  • Two hour serum or plasma glucose $ 200mg/dL during a 75 g oral glucose tolerance test (using WHO guidelines)

Obtaining any of the above results requires a repeat test on a different day to confirm the diagnosis.

The 1997 guidelines also recognize another group of patients who are defined as impaired fasting glucose or impaired glucose tolerance, depending upon the test which was performed. These patients will have a fasting glucose of 110 - 125 mg/dL or a 2-hour (OGTT) glucose of 140 - 199 mg/dL. Patients in this group are at a much greater risk of becoming diabetic than normal patients. Approximately 1-5% of these patients will become diabetic each year.

Once a diagnosis has been confirmed, laboratory testing is used to monitor the patient for the effectiveness of the treatment and compliance with prescribed care. The physician will order regular blood glucose testing and at specific intervals special tests for long term control will be performed. Tests to monitor long term control include glycohemoglobin, fructosamine, glycoalbumin, and microalbuminuria. The methods available and clinical use of these assays are described below:

Glycohemoglobin C. The assays to determine glycohemoglobin were developed to monitor diabetic patient glucose management over a 6 - 12 week time period. This test is tied to the life span of the red blood cells. Due to the frequent fluctuation of plasma glucose levels, this test reflects a more stable monitor of the therapeutic patient control. Hemoglobin A1c is one type of glycohemoglobin and is the one most commonly monitored. The 1997 ADA guidelines state that the hemoglobin A1c level should be less than 7% in patients exhibiting adequate control.

The glycohemoglobin molecule forms in two steps. In the first step, the aldehyde group on the glucose molecule reacts spontaneously and nonenzymatically with the amine group on the hemoglobin molecule to form a Schiff base. This reaction is reversible. In the second step of the reaction, the Schiff base undergoes a special biochemical rearrangement to form a ketoamine which is irreversible and yields a stable end product. In the presence of long term elevated serum glucose levels, the equilibrium of the above reaction favors the production of this ketoamine.

Glycohemoglobin assays are required to meet certain performance criteria to establish the method as acceptable. The four characteristics are (1) accuracy - the reported results must be standardized to hemoglobin A1c; (2) precision - National Institutes of Health (NIH) recommends that difference in %CV between run should be less than 5%; (3) the assay must be free from interference by labile aldemines, and (4) ideally, the assay should not be affected by variant hemoglobins.

HPLC and electrophoresis assays may show interference from the hemoglobin variants S and C since they appear as distinct bands that may migrate similarly with hemoglobin A. When these bands are present, hemoglobin A1c level may be falsely decreased . Hemoglobin F migrates in the same area as hemoglobin A1c and may exert a negative influence on hemoglobin A1c and trigger a decreased value.

Boronate affinity methods detect all forms of glycated hemoglobin. The correlation between hemoglobin A1c and total glycated hemoglobin is so close that they provide equivalent clinical information. As a result of this close correlation between the two analytes, assays for glycated hemoglobin may be standardized to hemoglobin A1c. In most cases this standardization of reporting is usually done by the instrument which is performing the test.

Fructosamine C. The name of this test is misleading. The test has nothing to do with fructose. When glucose attaches to a molecule of protein, the biochemical structure resembles a fructose molecule. The more appropriate name for this test would be glycoprotein or glycated protein. The measurement of glycated protein is another index of time-averaged plasma glucose. Since the serum proteins respond to change more quickly than hemoglobin, this assay is proposed to be more useful in monitoring the diabetic control over a 1-3 week period. The assay is not influenced by variant hemoglobin and is not attached to the lifespan of a red blood cell.

Fructosamine assays are based on the reduction of nitroblue tetrazolium (NBT) from blue to a purple color. Although many substances can reduce NBT, none of them are found in serum in high enough concentrations to cause significant interference with the assay. This assay is falsely elevated in patients with renal failure or liver disease with icterus or lipemia in the serum. The presence of hemolysis in the serum may cause a falsely decreased level. One final factor that limits the widespread use of this assay has to do with the difficulty in standardization of the test.

Glycoalbumin C. Although rarely used, this assay has been proposed to monitor diabetic control over a 1-2 week period. The test is an alternative to fructosamine, but also suffers from standardization problems.

One major area of concern when dealing with the diabetic patient is kidney function testing. Approximately 35% of the Type 1 diabetics and 15-60% of the Type 2 diabetics will develop diabetic nephropathy. Urine microalbumin is one of the best analytes for detection of diabetic nephropathy at an early reversible stage.

Urine Microalbumin C. The low concentration of albumin in urine is designated Amicroalbuminuria.@ Albumin occurs in the urine when there have been changes in the glomerular basement membrane which allows the filtration of albumin into the renal tubules creating a condition known as selective proteinuria. The damaged glomerular basement membrane will allow more plasma to flow into the renal tubules and thus cause elevated albumin levels. The presence of 30 - 300 mg/day of albumin excreted into the urine is defined as microalbuminuria. This level of excretion is below the detectable limit for total protein on a routine dipstick. Standard urine dipstick protein is positive in the presence of bacteria, WBC=s, and/or blood. A positive total protein in the absence of these elements indicates that the damage to the kidney is usually irreversible. The detection of an increased glomerular filtration rate is a reversible stage of diabetic nephropathy.

Current ADA recommendations include annual screening for microalbuminuria by: (1) albumin to creatinine ratio in random urine, (2) 24-hour urinary albumin excretion, or (3) another timed excretion of urinary albumin. Newer dipsticks and instrumentation can provide a rapid screen for the presence of microalbuminuria. Since microalbuminuria precedes irreversible kidney damage by 8-15 years, the screening tests for the albumin to creatinine ratio or microalbumin on random urine are very practical.

Esoteric Testing and Possible Clinical Uses

Detection of Autoantibodies in Type 1 Diabetic Mellitus C. 85 - 90% of the Type 1 diabetics will have at least one or more of the following autoantibodies present at the time of diagnosis: islet cell, insulin, glutamic acid decarboxylase, and tyrosine phosphatase. To date attempts to use this information for early detection of DM have been unsuccessful. Presently there are no clinical indications or benefits of these tests in early detection or prediction of DM.

Esoteric tests of glucose metabolism C. Other esoteric tests involve testing for insulin, C-peptide, glucagon and catecholamines. It has been found that insulin is elevated in the early stages of Type 2 diabetes. C-peptide has been found useful as an index of endogenous insulin production in patients being treated with exogenous insulin. C-peptide has been particularly useful for testing psychiatric patients who often have an artificial hypoglycemia. The relationship of glucagon and catecholamines is being evaluated in conjunction with insulin production.

Summary CAs tests to diagnose the disease and monitor diabetic care expand and become more intricate, the clinical laboratory will continue to stay on the cutting edge by providing tests that will support the treatment and care of these patients. The new ADA guidelines assist with early detection of the disease, thus protecting a patient from some of the long term complications. Physicians can monitor patient compliance with prescribed treatment through tests like hemoglobin A1c and fructosimine. Microalbuminuria testing provides a measure of the disease affect on the kidney. These recent advances allow the laboratory to providea means to better patient care. From the early diagnosis with the oral glucose tolerance test to present day monitoring of patient compliance with hemoglobin A 1c, the clinical lab is a vital component for patient care and management.

References

Kaplan, L and Pesce, A, Clinical Chemistry theory, analysis, and correlation, St. Louis, MO, 1984, C.V. Mosby

The Clinical Chemistry of Diabetes, Workshop, Washington, DC, October,1998

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