Introduction

A glucose tolerance test (GTT) is a procedure that determines whether a patient can use and store glucose normally.[1] The test is usually used to test for diabetes mellitus, insulin resistance, impaired pancreatic beta cell function, and sometimes reactive hypoglycemia or acromegaly, or rarer disorders of carbohydrate metabolism.[2] In the most commonly performed version of the test, an oral glucose tolerance test (OGTT), a standard dose of glucose is ingested by mouth, and blood levels are checked two hours later.[3] Many variations of the GTT have been devised over the years for various purposes, with different standard doses of glucose, different administration routes, different sampling intervals, and various substances measured in addition to blood glucose.[1]

Etiology and Epidemiology

Diabetes mellitus is a group of metabolic disorders characterized by hyperglycemia resulting from defects in insulin secretion, insulin action, or both.[4] The latest data from the Centers for Disease Control and Prevention indicate that nearly 37.3 million Americans with diabetes and almost 96 million people aged 18 years or older have prediabetes (38.0% of the adult U.S. population). Approximately 90% to 95% of all U.S. diabetes cases are type 2.[5] Type 2 diabetes is estimated to be undiagnosed in at least 30% of the U.S. population.[6]

Type 1 diabetes mellitus results from cellular-mediated autoimmune destruction of the insulin-secreting cells of pancreatic beta cells.[7] In the vast majority of patients, destruction is mediated by T cells.[8] The autoimmune process leading to type 1 diabetes begins months or years before the clinical presentation. An 80% to 90% reduction in the volume of beta cells is required to induce symptomatic type 1 diabetes.[9] The rate of islet cell destruction is variable and is usually more rapid in children than adults.[10]

Type 2 diabetes mellitus accounts for approximately 90% of all cases of diabetes. Patients have minimal symptoms, are not prone to ketosis, and are not dependent on insulin to prevent ketonuria.[11] Insulin concentrations may be normal, decreased, or increased, and most people with this form of diabetes have impaired insulin action.[12] Obesity is a commonly associated condition, and weight loss alone may improve hyperglycemia in these persons. However, many individuals with type 2 diabetes may require dietary intervention, antihyperglycemic agents, or insulin to control hyperglycemia.[13] Most patients acquire the disease after age 40, but it may occur in younger people.[14]

Gestational diabetes mellitus (GDM) is defined as any degree of glucose intolerance (i.e., hyperglycemia) with onset or first recognition during pregnancy (i.e., patients who become pregnant after a diagnosis of diabetes mellitus are not included in this category).[15] The estimated frequency of abnormal glucose tolerance during pregnancy ranges from less than 1% to 28%, depending on the population studied and the diagnostic tests employed. The prevalence of GDM is increasing, at least partly, due to the considerable increase in obesity.[16]

Patients with GDM are at significantly greater risk for the subsequent development of type 2 diabetes mellitus, which occurs in 15% to 60%. The risk is exceptionally high in patients with marked hyperglycemia during or soon after pregnancy, patients who are obese, and patients whose GDM is diagnosed before 24 weeks gestation.[17] At 6 to 12 weeks postpartum, all patients who had GDM should be evaluated for diabetes using nonpregnant OGTT criteria. If diabetes is not present, patients should be re-evaluated for diabetes at least every three years.[18]

Pathophysiology

Insulin resistance and an insulin secretory deficiency brought on by beta cell dysfunction are two defects that characterize the transition from normal glucose tolerance to type 2 diabetes.[19] Reduced tissue sensitivity to insulin and pronounced compensatory hyperinsulinemia are symptoms of insulin resistance.[20] Plasma glucose levels are kept within the usual range at first. In patients who will eventually develop diabetes, there is a decline in beta cell secretory capacity.[13] The first glucose abnormality detected is a rise in the postprandial glucose levels because of reduced first-phase insulin secretion.[11] The fasting glucose levels rise as beta cell activity deteriorates over time. Diabetes eventually develops with a further reduction of insulin secretion.[12]

Specimen Requirements and Procedure

The choice of specimen used for glucose determination depends on the analytical method to be used. Serum or plasma, free of hemolysis, is the specimen of choice for automated enzymatic methods.[21] However, the glucose concentration in whole blood is approximately 12% to 15% lower than in the plasma because of the higher water content.[22] Plasma is recommended for the diagnosis of diabetes since the diagnostic cutoff points have been derived using plasma samples. Glucose concentrations in heparinized plasma are approximately 5% lower than in serum.[23]

The plasma should be separated from the cells within 60 minutes of collection unless the tube contains a glycolysis inhibitor. Serum samples are appropriate for glucose analysis, provided the serum is not in contact with the cells for longer than 90 minutes.[24] Glucose in whole blood at room temperature can undergo glycolysis at a rate of approximately 5% to 7% (10 mg/dL or 0.6 mmol/L per hour).[22] The sample should be centrifuged and removed from clots or cells as soon as possible. In patients with leukocytosis or samples contaminated with bacteria, the rate of glycolysis can be even higher.[23]

To preserve blood that cannot be separated rapidly, samples should contain the glycolysis inhibitor sodium fluoride (which inhibits the enolase enzyme) at 2.5 mg fluoride/mL of blood.[25] This reagent is used in combination with anticoagulants such as potassium oxalate. The glucose concentrations are stable for 72 hours at room temperature with fluoride, but there is a 10% drop in the concentration due to a water shift from the cells.[23] The glucose concentration does not change in the serum or plasma samples collected in tubes that contain gel separators. The glucose in these tubes is stable for at least one week when stored at 4^°C.[26]

Diagnostic Tests

Plasma glucose can be measured with high precision and accuracy using enzymatic methods such as glucose oxidase and hexokinase. The hexokinase method is considered the fastest and most accurate and is commonly used on automated systems.[27] In glucose hexokinase, the reaction uses glucose-6-phosphate dehydrogenase and yields precise results.[28] Generally, fasting plasma glucose, 2 hours post glucose during a 75-gram OGTT, and A1C are equally appropriate for diagnostic screening.[29] It should be noted that the detection rates of different screening tests vary in both populations and individuals.[20] Moreover, the efficacy of interventions for the primary prevention of type 2 diabetes has mainly been demonstrated among individuals who have impaired glucose tolerance (IGT) with or without elevated fasting glucose, not for individuals with isolated impaired fasting glucose (IFG) or for those with prediabetes defined by A1C criteria.[19]

Testing Procedures

Serial measurement of plasma glucose before and after a specific amount of glucose given orally should provide a standard method to evaluate individuals and establish values for healthy and diseased subjects.[1] Although more sensitive than fasting plasma glucose determinations, glucose tolerance testing is affected by multiple factors that result in poor reproducibility.[30] Moreover, approximately 20% of OGTTs fall into the non-diagnostic category (e.g., only one blood sample exhibits increased glucose concentration). Unless results are grossly abnormal initially, the OGTT should be performed on two separate occasions to establish the diagnosis of diabetes.[31]

The following conditions should be met before an OGTT is performed: discontinue, when possible, medications known to affect glucose tolerance; perform the test in the morning after three days of unrestricted diet (containing at least 150 g of carbohydrate per day) and activity; and perform the test after a 10- to 16-hour fast only in ambulatory outpatients (bed rest impairs glucose tolerance), who should remain seated during the test without smoking cigarettes. Glucose tolerance testing should not be performed on hospitalized, acutely ill, or inactive patients. The test should begin between 7:00 a.m. and 9:00 a.m. Venous plasma glucose should be measured fasting and two hours after an oral glucose load. For nonpregnant adults, the recommended load is 75 g, which may not be a maximum stimulus; for children, 1.75 g/kg, up to a maximum of 75 g, is given.[1] The glucose should be dissolved in 250 to 300 mL of water and ingested over 5 minutes. A commercial, more palatable form of glucose may be ingested, but whether the anhydrous or monohydrate form of glucose should be used is still in question.[31]

Human growth hormone (hGH) suppression by a glucose load is a classic screening test for acromegaly.[32] The following conditions should be met before an OGTT is performed for acromegaly - the patient should be fasting before the start of the test. Blood is drawn for a baseline serum growth hormone level. A drink containing 75 grams of glucose is administered. Samples for analysis are collected for serum growth hormone every 30 minutes for two hours at 30-, 60-, 90-, and 120-minute intervals after ingesting the glucose drink. Centrifuge the samples within one hour after the blood is drawn and label the specimens as a baseline, 30, 60, 90, and 120 minutes.[33]

Interfering Factors

Several precautions must be taken in preparing for and performing the oral glucose tolerance test. The OGTT should not be performed on patients suffering from an intercurrent infection or the effects of trauma or those recovering from a severe illness.[1] Drugs such as corticosteroids and diuretics may impair glucose tolerance and should be stopped before the test if possible. The patient should have been on an unrestricted diet containing at least 150 g of carbohydrates daily for at least three days and not indulged in unaccustomed amounts of exercise.[30]

Results, Reporting, and Critical Findings

Impaired fasting glucose (IFG) is defined as fasting plasma glucose (FPG) levels from 100 to 125 mg/dL (from 5.6 to 6.9 mmol/L) and impaired glucose tolerance as two-hour post glucose levels during 75-gram OGTT from 140 to 199 mg/dL (from 7.8 to 11.0 mmol/L).[34] It should be noted that the World Health Organization and numerous other diabetes organizations define the IFG lower limit at 110 mg/dL (6.1 mmol/L).[35]

The results of the OGTT as a screening test for type 2 diabetes can be interpreted as follows:

• The 2-hour plasma glucose level <140 mg/dL is considered normal
• The 2-hour plasma glucose level of 140-199 mg/dL indicates impaired glucose tolerance
• The 2-hour plasma glucose level ≥200 mg/dL indicates diabetes

For a diagnosis to be made, the test must be repeated on another day shortly afterward, yielding similar results. Alternatively, a diagnosis can be confirmed using one of the other screening tests. A single abnormal OGTT is insufficient for diagnosing diabetes or prediabetes.[29]

The American Diabetes Association has recommended using either the one- or two-step approach at 24–28 weeks of gestation in pregnant patients not previously known to have diabetes.[36]

One-Step Strategy for Screening  and Diagnosis of GDM

The diagnosis of GDM is made when any of the following plasma glucose values are met or exceeded during one step strategy:

• Fasting plasma glucose: 92 mg/dL (5.1 mmol/L) 
• 1-h plasma glucose: 180 mg/dL (10.0 mmol/L)
• 2-h plasma glucose: 153 mg/dL (8.5 mmol/L)

Two-Step Strategy for Screening and Diagnosis of GDM

If the plasma glucose level measured one hour after the 50-gram glucose load, at 24–28 weeks of gestation in individuals not previously diagnosed with diabetes, is more than or equal to 130, 135, or 140 mg/dL (7.2, 7.5, or 7.8 mmol/L, respectively), proceed to a 100-gram OGTT. The 100-gram OGTT should be performed when the patient is fasting.

The diagnosis of GDM is made when at least two of the following four plasma glucose levels (measured fasting and at 1, 2, and 3 hours during OGTT) are met or exceeded: 

• Fasting plasma glucose: 95 mg/dL (5.3 mmol/L) 
• 1-h plasma glucose: 180 mg/dL (10.0 mmol/L) 
• 2-h plasma glucose: 155 mg/dL (8.6 mmol/L)
• 3-h plasma glucose: 140 mg/dL (7.8 mmol/L)

Oral Glucose Tolerance Test in Acromegaly 

The diagnostic criteria for acromegaly are met if the growth hormone level does not suppress below 1 ng/mL. The suppression test is reported to have a false-negative rate as high as 50%.[33] The sensitivity of the test is reported to be improved at a cutoff of 0.4 ng/mL. The diagnosis includes clinical signs of growth hormone excess and elevated IGF-1 levels.[37] False-positive results with values that remain higher than 1 ng/mL after glucose administration may be observed during puberty and in patients with diabetes mellitus, liver disease, renal disease, or anorexia nervosa.[38]

Clinical Significance

The GTT only establishes the presence of glucose intolerance. It is used in patients with borderline fasting and postprandial glucose to support or rule out the diagnosis of diabetes mellitus. Some use it in unexplained hypertriglyceridemia, neuropathy, impotence, diabetes-like renal diseases, and retinopathy.[1] The OGTT is used to work up glycosuria without hyperglycemia (e.g., renal glycosuria). It is used to predict perinatal morbidity in pregnancy and to diagnose gestational diabetes.[30] The risks of fetal abnormality and perinatal mortality increase with abnormal carbohydrate metabolism in pregnancy.[31]

Reactive or postprandial hypoglycemia causes blood glucose to decrease two to five hours after a diet with high carbohydrate content. Early and late postprandial hypoglycemia occurs 2 to 3 hours and 3 to 5 hours after a meal, respectively.[39] A five-hour oral glucose tolerance test (5-OGTT) is often a helpful laboratory investigation to understand and establish a diagnosis of postprandial hypoglycemia. However, in clinical practice, the use of a 5-OGTT is discredited for diagnosing reactive hypoglycemia as it may show false-positive results.[40]

Quality Control and Lab Safety

For non-waived tests, laboratory regulations require, at the minimum, analysis of at least two levels of control materials once every 24 hours. Laboratories can assay QC samples more frequently to ensure accurate results.[41] Quality control samples should be assayed after calibration or maintenance of an analyzer to verify the correct method performance. To minimize QC when performing tests for which manufacturer recommendations are less than those required by the regulatory agency (such as once per month), the labs can develop an individualized quality control plan (IQCP) that involves performing a risk assessment of potential sources of error in all phases of testing and putting in place a QC plan to reduce the likelihood of errors.[42] Westgard multi-rules are used to evaluate the quality control runs. In case of any rule violation, proper corrective and preventive action should be taken before patient testing.[43]

The laboratory must participate in the external quality control or proficiency testing (PT) program because it is a regulatory requirement published by the Centers for Medicare and Medicaid Services (CMS) in the Clinical Laboratory Improvement Amendments (CLIA) regulations.[44] It is helpful to ensure the accuracy and reliability of the laboratory with regard to other laboratories performing the same or comparable assays. The required participation and scored results are monitored by CMS and voluntary accreditation organizations.[45] The PT plan should be included as an aspect of the quality assessment (QA) plan and the overall quality program of the laboratory.[46]

The criteria for acceptable performance for glucose assay by the Clinical Laboratory Improvement Amendments (CLIA) and College of American Pathologists (CAP) proficiency program is within ± 6mg/dL or ± 10% of the mean value of laboratory peer groups.[47]

All specimens, control materials, and calibrator materials should be considered potentially infectious. Exercise the normal precautions required for handling all laboratory reagents. Disposal of all waste material should be in accordance with local guidelines. Wear gloves, a lab coat, and safety glasses when handling human blood specimens.[48] Place all plastic tips, sample cups, and gloves that come into contact with blood in a biohazard waste container. Discard all disposable glassware into sharps waste containers. Protect all work surfaces with disposable absorbent bench top paper, and discard it into biohazard waste containers weekly or whenever blood contamination occurs. Wipe all work surfaces weekly.[49]

Enhancing Healthcare Team Outcomes

A glucose tolerance test is typically ordered by a medical doctor, advanced nurse practitioner, or physician assistant. Interprofessional collaboration is required for the correct administration of the test.[50] The provider or the nurse must give the patient adequate instructions to prepare for the examination and what to expect during the test.[51]

The actual glucose tolerance test itself can be administered in several settings. The test may be administered in a clinical office with appropriate equipment and staffing. The glucose tolerance test can also be administered in a laboratory. Although the inpatient side is an atypical site for glucose tolerance tests, a hospital may have an outpatient or clinical research department where staff may have additional time to complete the test.

Nurses, medical assistants, or phlebotomists may perform the test. There must be clear communication on order from the provider on the type of test, the length of time, and the number of samples ordered. The personnel administering the test should be aware of the requirements of the test, including the fasting requirement and the pre-test dietary carbohydrate requirement. It is vital to collaborate with laboratory personnel to ensure the timely processing of the specimens and proper storage and shipping (if necessary).

Laboratory personnel should work closely with providers to provide accurate results quickly.