Glucose Tolerance Test

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 have diabetes, and almost 96 million people aged 18 years or older—38.0% of the adult U.S. population—have prediabetes. Approximately 90% to 95% of all U.S. diabetes cases are type 2.[5] Type 2 diabetes mellitus (T2DM) is estimated to be undiagnosed in at least 30% of the U.S. population.[6]

Type 1 diabetes mellitus (T1DM) results from cell-mediated autoimmune destruction of the insulin-secreting β cells of the pancreas.[7] Destruction is mediated by T cells in the vast majority of patients.[8] The autoimmune process leading to T1DM begins months or years before clinical presentation. An 80% to 90% reduction in β-cell volume is required to induce symptomatic T1DM.[9] The rate of islet cell destruction is variable and usually more rapid in children than in adults.[10]

T2DM accounts for approximately 90% of all diabetes cases. 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 T2DM have impaired insulin action.[12] Obesity is commonly associated, and weight loss alone may improve hyperglycemia. However, many individuals with T2DM require dietary intervention, antihyperglycemic agents, or insulin to control blood glucose levels.[13] Most patients develop the disease after age 40, although it may occur in younger people.[14]

Gestational diabetes mellitus (GDM) is defined as any degree of glucose intolerance (ie, hyperglycemia) with onset or first recognition during pregnancy. 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 rise in obesity.[16]

Patients with GDM are at significantly higher risk of developing T2DM, occurring in 15% to 60% of cases. The risk is especially high in individuals with obesity, marked hyperglycemia during or soon after pregnancy, or GDM diagnosed before 24 weeks of gestation.[17] All patients who had GDM should be evaluated for diabetes using nonpregnant OGTT criteria at 6 to 12 weeks postpartum. These individuals should be reevaluated at least every 3 years if diabetes is not detected at this time.[18]

Pathophysiology

Insulin resistance and an insulin secretory deficiency induced by β-cell dysfunction are the 2 defects that characterize the transition from normal glucose tolerance to T2DM.[19] Reduced tissue sensitivity to insulin and pronounced compensatory hyperinsulinemia are features of insulin resistance.[20] Plasma glucose levels remain within the normal range initially. β-cell secretory capacity declines in patients who eventually develop T2DM. The first detectable glucose abnormality is a rise in postprandial glucose levels due to reduced 1st-phase insulin secretion. Fasting glucose levels increase as β-cell activity deteriorates over time, and diabetes eventually develops with further reduction of insulin secretion.

Specimen Requirements and Procedure

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

Plasma should be separated from cells within 60 minutes of collection unless the tube contains a glycolysis inhibitor. Serum samples are suitable for glucose analysis, provided they are not in contact with cells for longer than 90 minutes.[24] Glucose in whole blood at room temperature undergoes glycolysis at a rate of approximately 5% to 7% (10 mg/dL or 0.6 mmol/L) per hour. Samples should be centrifuged and separated from clots or cells as soon as possible. The rate of glycolysis can be even higher in individuals with leukocytosis or bacterial contamination.

To preserve blood that cannot be separated rapidly, samples should contain the glycolysis inhibitor sodium fluoride, which inhibits enolase, at 2.5 mg fluoride/mL of blood.[25] This reagent is used with anticoagulants such as potassium oxalate. Glucose concentrations remain stable for 72 hours at room temperature with fluoride, although glucose decreases by 10% due to water shift from the cells. Glucose concentration does not change in serum or plasma samples collected in tubes with gel separators. Glucose is stable for at least 1 week when stored at 4 °C in these tubes.[26]

Diagnostic Tests

Plasma glucose may be measured with high precision and accuracy using techniques involving enzymatic reagents, particularly glucose oxidase and hexokinase. The hexokinase method is considered the fastest and most accurate and is commonly used on automated systems.[27] Hexokinase irreversibly phosphorylates glucose to glucose-6-phosphate, which is then coupled to glucose-6-phosphate dehydrogenase activity, enabling highly specific and sensitive measurement as well as metabolic control.[28] By comparison, glucose oxidase methods are more susceptible to interference from substances such as uric acid and ascorbic acid.

Fasting plasma glucose (FPG), glycated hemoglobin A1C (HbA1C), and 2-hour post-glucose load measurement during a 75-gram OGTT are all suitable for diabetes diagnostic screening.[29] Detection rates of different screening tests vary across populations and individuals. The efficacy of interventions for primary prevention of T2DM has mainly been demonstrated in people with impaired glucose tolerance (IGT), with or without elevated fasting glucose, rather than in individuals with isolated impaired fasting glucose (IFG) or prediabetes according to HbA1C thresholds.

Testing Procedures

Serial measurement of plasma glucose before and after a specific oral glucose load provides a standard method to evaluate individuals and establish values for healthy and diseased subjects. Although more sensitive than FPG determinations, GTT is influenced by multiple factors, resulting in poor reproducibility.[30] Approximately 20% of OGTTs fall into the nondiagnostic category (eg, only 1 blood sample exhibits increased glucose concentration). Unless results are grossly abnormal initially, the OGTT should be performed on 2 separate occasions to establish a diabetes diagnosis.

Conditions for performing an OGTT include the following:

• Medications that affect glucose tolerance should be discontinued when possible.
• The test must be conducted in the morning after 3 days of unrestricted diet (≥150 g carbohydrate per day) and normal activity.
• The test should be performed after a 10- to 16-hour fast in ambulatory outpatients. Patients must remain seated, and smoking should be avoided.
• Glucose tolerance is impaired by bed rest. The evaluation should not be performed in hospitalized, acutely ill, or inactive patients.
• The test must begin between 7:00 a.m. and 9:00 a.m.
• Venous plasma glucose should be measured fasting and 2 hours after the oral glucose load.
• A 75-g glucose load must be administered to nonpregnant adults. Children should receive 1.75 g/kg, up to a maximum of 75 g.
• Glucose should be dissolved in 250 to 300 mL of water and ingested over 5 minutes.

A commercial, more palatable glucose formulation may be ingested. Whether the anhydrous or monohydrate form is used remains uncertain.[31]

Human growth hormone suppression by a glucose load is a classic screening test for acromegaly.[32] Patients being evaluated for this condition must fast before starting an OGTT. A baseline blood sample is drawn for measurement of serum growth hormone. A drink containing 75 grams of glucose is administered. Blood samples for serum growth hormone analysis are collected at 30, 60, 90, and 120 minutes after glucose ingestion. Samples should be centrifuged within 1 hour of collection and labeled according to the time points: baseline, 30, 60, 90, and 120 minutes.[33]

Interfering Factors

Several precautions must be observed when preparing for and performing the OGTT. The assessment should not be conducted in patients with an infection, traumatic injuries, or severe illness. Drugs such as corticosteroids and diuretics, which may impair glucose tolerance, must be discontinued before the test if possible. The patient should have followed an unrestricted diet containing at least 150 g of carbohydrates daily for at least 3 days and avoided unusual or vigorous exercise during that period.

Results, Reporting, and Critical Findings

IFG is defined as having FPG levels of 100 to 125 mg/dL (5.6 to 6.9 mmol/L). IGT is characterized by having 2-hour postglucose load levels ranging from 140 to 199 mg/dL (7.8 to 11.0 mmol/L) during a 75-gram OGTT.[34] The World Health Organization and several other diabetes organizations define the lower IFG limit as 110 mg/dL (6.1 mmol/L).[35]

The results of the OGTT as a screening test for T2DM may be interpreted as follows:

• 2-hr plasma glucose below 140 mg/dL: normal
• 2-hr plasma glucose ranging from 140 to 199 mg/dL: IGT
• 2-hr plasma glucose of at least 200 mg/dL: diabetes

Confirming a diagnosis requires repeating the test on another day shortly afterward and yielding similar results. Alternatively, a diagnosis may be established using one of the other recommended screening tests. A single abnormal OGTT is insufficient to diagnose diabetes or prediabetes.

The American Diabetes Association recommends either the 1- or 2-step approach at 24 to 28 weeks of gestation for pregnant patients not previously known to have diabetes.[36]

One-Step Strategy for Screening  and Diagnosis of Gestational Diabetes Mellitus

The diagnosis of GDM is made when any of the following plasma glucose values are met or exceeded during a 1-step strategy:

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

This method entails administering a 75-gram glucose drink to a fasting patient and obtaining plasma glucose readings at baseline, 1 hour, and 2 hours postload. The 1-step OGTT is highly sensitive for diagnosing GDM, capturing postprandial glucose abnormalities missed by simpler tests. 

Two-Step Strategy for Screening and Diagnosis of Gestational Diabetes Mellitus

A 100-gram OGTT is recommended if plasma glucose at 1 hour after a 50-g glucose load, performed at 24 to 28 weeks of gestation in those not previously diagnosed with diabetes, reaches or exceeds threshold values of 130, 135, or 140 mg/dL (7.2, 7.5, or 7.8 mmol/L, respectively). The assessment should be conducted when the patient is fasting.

A diagnosis of GDM is established if at least 2 out of 4 plasma glucose measurements taken fasting and at 1, 2, and 3 hours during the OGTT meet or exceed the following.

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

By evaluating glucose tolerance at fasting and several intervals after the glucose load, the 100-gram OGTT provides a detailed metabolic profile critical for accurate GDM diagnosis. However, the complexity of this method has spurred investigation into alternative screening strategies that balance precision with patient convenience.

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. However, the growth hormone suppression test has been reported to have a false-negative rate of up to 50%. Sensitivity improves when a cutoff of 0.4 ng/mL is used. Diagnosis requires clinical signs of growth hormone excess and elevated insulin-like growth factor 1 levels.[37] False-positive results where the hormone remains above 1 ng/mL after glucose administration may occur in individuals going through puberty or experiencing diabetes mellitus, liver disease, renal disease, or anorexia nervosa.[38]

Clinical Significance

The GTT establishes the presence of glucose intolerance. This diagnostic tool is indicated in patients with borderline fasting or postprandial glucose to support or rule out the diagnosis of diabetes mellitus. The test may also be applied in cases of unexplained hypertriglyceridemia, neuropathy, impotence, diabetes-like renal disease, and retinopathy. The OGTT is used to evaluate glycosuria without hyperglycemia (eg, renal glycosuria), predict perinatal morbidity during pregnancy, and diagnose GDM. Abnormal carbohydrate metabolism in pregnancy increases the risk of fetal abnormalities and perinatal mortality.

Reactive, or postprandial, hypoglycemia is characterized by a decrease in blood glucose 2 to 5 hours after a high-carbohydrate meal. Early postprandial hypoglycemia occurs 2 to 3 hours after a meal, and late postprandial hypoglycemia occurs 3 to 5 hours afterward.[39] A 5-hour OGTT (5-OGTT) may be a useful laboratory investigation to evaluate postprandial hypoglycemia. However, in clinical practice, the 5-OGTT is generally not recommended for diagnosing reactive hypoglycemia due to the risk of false-positive results.[40]

Quality Control and Lab Safety

The implementation of a robust quality management system is essential in clinical laboratories that perform patient testing, including the OGTT. Quality management systems ensure the accuracy, reliability, and consistency of test results by integrating quality assurance practices across all phases of testing. In the context of the OGTT, standardization of the preanalytical phase is particularly critical, as patient preparation significantly influences the diagnostic outcome. Clear protocols regarding dietary intake, fasting duration, and physical activity must be strictly followed to minimize variability and ensure clinical validity. In the analytical phase, quality is maintained through internal quality control and external quality assessment, both of which are essential for monitoring performance and ensuring result validity.

For nonwaived tests, laboratory regulations require analysis of at least 2 levels of control materials once every 24 hours. Laboratories may assay quality control samples more frequently to ensure accurate results.[41] Quality control samples should also be assayed after calibration or maintenance of an analyzer to verify correct method performance.

When manufacturer quality control recommendations are lower than regulatory requirements (eg, once per month), laboratories can implement an individualized quality control plan. This strategy involves performing a risk assessment of potential sources of error across all phases of testing and establishing a quality control protocol to reduce the likelihood of errors.[42] Westgard multirules are applied to evaluate quality control runs, and any rule violations require appropriate corrective and preventive actions before patient testing.[43]

The laboratory must participate in an external quality control or proficiency testing program, as required by the Centers for Medicare and Medicaid Services (CMS) under the Clinical Laboratory Improvement Amendments (CLIA) regulations.[44] Participation helps ensure the accuracy and reliability of laboratory results compared with other laboratories performing the same or comparable assays. Required participation and scored results are monitored by the CMS and voluntary accreditation organizations.[45] The proficiency testing plan should be incorporated into the laboratory’s quality assessment plan and overall quality program.[46] According to CLIA and the College of American Pathologists' proficiency program, glucose assay results are acceptable if they deviate no more than 6 mg/dL or 8% from the mean value of laboratory peer groups.[47]

All specimens, control materials, and calibrators should be treated as potentially infectious. Standard precautions for handling laboratory reagents should be followed. Waste disposal must comply with local guidelines. Personal protective equipment, including gloves, a lab coat, and safety glasses, should be worn when handling human blood specimens.[48] Plastic tips, sample cups, and gloves that contact blood should be discarded in a biohazard waste container, and disposable glassware should be placed in sharps waste containers. Work surfaces should be protected with disposable absorbent bench-top paper, which should be discarded into biohazard waste containers weekly or whenever contamination occurs. All work surfaces should be wiped weekly.[49]

Enhancing Healthcare Team Outcomes

A GTT is typically ordered by a medical doctor, advanced practice nurse, or physician assistant. Interprofessional collaboration is required to ensure correct test performance.[50] The healthcare provider or nurse must give the patient clear instructions on how to prepare for the examination and what to expect during the test.[51]

The GTT may be administered in several settings. The evaluation may be performed in a clinical office, equipped with the necessary instruments and staffing, or a laboratory. Although inpatient units are an atypical setting for the task, a hospital’s outpatient or clinical research department may provide staff with additional time to complete this examination.

Nurses, medical assistants, or phlebotomists may perform the test. Clear communication of the healthcare provider’s order is essential, including the type of test, duration, and number of samples required. Personnel administering the test must be aware of all test requirements, including fasting and pretest dietary carbohydrate intake. Collaboration with laboratory staff is vital to ensure timely specimen processing, proper storage, and shipping if necessary. Laboratory personnel should work closely with providers to deliver accurate results efficiently.