Peroxidase-Coupled Glucose Method

Specimen Requirements and Procedure

A blood sample is drawn into a sodium fluoride vial for testing. Sodium fluoride inhibits the glycolytic enzyme enolase present in red blood cells (RBCs), and thus its anti-glycolytic effects prevent a falsely low blood glucose value. Plasma is then obtained by centrifuging the blood sample for 10 minutes at 2000 rpm. When calculating blood glucose concentration, it is important to note that the concentration of glucose in whole blood is estimated to be 10 to 15% lower than the glucose concentration in plasma. The glucose concentration in the water content of the plasma is equal to the concentration of glucose in the water content of RBCs. The plasma, however, has greater water content and therefore has a greater glucose concentration.[5] It is important to obtain the glucose concentration from the plasma for a more accurate blood glucose reading.  

The materials required to analyze blood glucose levels using the GOD-POD method are as follows:

• Sample of blood in sodium fluoride vial
• Glucose reagent containing glucose oxidase, peroxidase, 4-amino antipyrine, and phenol
• Glucose Standard containing a known glucose concentration (100 mg/dL)
• Constant temperature incubator set at 37 degrees Celcius 
• Pipettes for sample measuring 
• Dry test tubes
• Colorimeter capable of measuring absorbance between 500nm to 520 nm

Procedure 

Step 1: Take three test tubes and label each as Blank, Standard, and Test 

Step 2: Add 10 microliters of distilled water to a blank test tube

Step 3: Add 10 microliters of glucose standard (control) to a standard test tube

Step 4: Add 10 microliters of the plasma being analyzed in a test tube

Step 5: Add 1 ml of glucose reagent to each test tube

Step 6: Mix each tube thoroughly and incubate for 10-15 minutes at 37 degrees Celsius

Step 7: Measure the optical density of each sample using the colorimeter set at 505 nm 

Testing Procedures

Enzymatic methods are the most widely used methods for glucose analysis; however, other methods for glucose quantification include reducing and condensation methods. The least specific of these methods is the reducing method. This technique utilizes the reducing ability of glucose to reduce metals, such as copper, which yields a color change in the solution. The downfall of this method is that numerous reducing agents can interfere with the estimation of glucose and produce a significantly falsely elevated result.[3]

Condensation methods utilize the aldehyde group of glucose, which can react with aromatic compounds, such as o-toluidine, to form glucosamine that produces a green color. The color is then measured to yield an estimation of the glucose concentration in the sample. However, other aldoses can cross-react and cause inaccurate results.[3]

Enzymatic methods, like the GOD-POD and hexokinase methods, are the most widely used methods for glucose estimation in the laboratory. The hexokinase method, in particular, is a highly specific modality for determining plasma glucose. The enzymatic method yields NADH through hexokinase-catalyzed transformations of glucose. NADH is then analyzed using spectrophotometry to determine the glucose concentration in the sample.

Interfering Factors

High levels of reducing agents such as uric acid, vitamin C, and bilirubin may affect results. 4-aminoantipyrine, a component of the GOD-POD reaction, is an oxidizing agent that can neutralize excess reducing substances in the bloodstream and prevent interference with the coloring agent, phenol. Other potential interfering substances include excess triglycerides, abnormal hematocrit levels, and various drugs and medications.[6]

Results, Reporting, and Critical Findings

Generally, a normal fasting blood glucose range is between 70-99 mg/dL, a fasting blood glucose range of 100 to 125 mg/dL indicates pre-diabetes, and a fasting blood glucose of >125 mg/dL indicates diabetes. However, there are slight variations in reference ranges between different laboratories, and the reference values of the facility where the test was performed should be used. To calculate a patient's blood glucose level using the GOD-POD method, the optical density of the Test, Standard, and Blank are taken. 

(OD of Test- OD blank) x Conc. Standard) / (OD of Standard - OD blank)

The optical density of the blank should equal zero, and thus the formula is simplified to:

(OD of Test x Conc. Standard) / OD of Standard

The optical density of the test is divided by the optical density of the standard, and the value obtained is then multiplied by the known glucose concentration of the mean (100mg/dL). The value obtained from this calculation is the glucose concentration of the test sample. 

Clinical Significance

Glucose is derived from the breakdown of carbohydrates, and its availability is essential for providing energy to the human body. The body tightly controls glucose within a normal, healthy range through endocrine hormones such as insulin and glucagon. Other hormones that can significantly affect glucose levels are cortisol, epinephrine, and thyroid hormone. When symptoms of abnormally controlled blood glucose bring a patient to seek medical attention, a blood glucose measurement is an essential aspect of data collection to determine the pathology and treatment of the condition.

Some physiological conditions associated with hyperglycemia include a high carbohydrate diet, anxiety, emotional stress, and pregnancy. Pathological conditions associated with hyperglycemia include hyperpituitarism, hyperthyroidism, hyperadrenalism, and diabetes mellitus. 

Diabetes mellitus can further be subdivided into type I and type II. Type I diabetes is due to autoimmune destruction of the beta-cells within the pancreas and tends to occur early in life. The beta-cells are responsible for insulin secretion; thus, in type 1 diabetes, there is an absolute insulin deficiency. Type II diabetes is more of a progressive loss of function of the beta-cells and is mainly due to insulin resistance. It typically occurs in adults with risk factors such as obesity, sedentary lifestyles, and a family history of the disease. Chronically elevated glucose levels can lead to microvascular damage within the kidneys, the eyes, and the peripheral nerves; this damage can manifest as nephropathy, retinopathy, and neuropathy. 

Hyperpituitarism, hyperthyroidism, and hyperadrenalism are disorders in which aberrant hormonal signaling within the body increases the breakdown of molecules into glucose or increases the production of glucose. Although the pathophysiology of these disorders is different than diabetes, one of their manifestations is abnormally elevated blood glucose, and thus accurate measurements of glucose levels aid in the clinical diagnosis.   

Hypoglycemia can also be physiological, as would be seen in starvation and after excessive, strenuous exercise. Pathological conditions associated with hypoglycemia include insulin overdose or an insulin-secreting tumor, hypothyroidism, hypopituitarism, and Addison disease. Excess insulin causes the body to uptake too much glucose from the blood and thus causes hypoglycemia. Hypothyroidism, hypopituitarism, and Addison disease are all disorders in which the body’s endocrine hormones that signal the body’s organs to increase blood glucose are lacking. Although other symptoms are associated with these diseases, hypoglycemia is a common and clinically relevant finding. 

Just as well-controlled blood glucose is essential for overall health, accurate glucose measurements are crucial for a clinician to provide the best care to a patient. 

Quality Control and Lab Safety

Using a blank test and a standard (or control) with a known glucose concentration allows for accurate results independent of the technical faults with the colorimeter or instruments used in the analysis. Each test performed on an unknown sample should include samples with normal and abnormal glucose concentrations to allow for appropriate quality control. If the results of the controls are not consistent with what was expected, be sure to check the procedure, the colorimeter, and the instruments used for the analysis. 

Enhancing Healthcare Team Outcomes

Interdisciplinary collaboration is essential when planning and managing a patient's medical condition. If a patient is hyperglycemic, laboratory studies are necessary for the clinician to properly treat the abnormally elevated blood glucose levels. Based on the lab's objective data, the clinician can then dose the insulin, fluids, and electrolytes to the patient's current status. The role of the nurse is essential in carrying out the clinician's orders of the insulin prescribed and carefully monitoring the patient's vital signs to ensure that the patient is responding appropriately. In addition to medical therapy, a registered dietitian can provide nutritional therapy to help patients manage their blood glucose and possibly prevent another hospitalization. As with all medical conditions, collaboration and communication between clinicians (MDs, DOs, NPs, and PAs), nurses, laboratory clinicians, and all other disciplines involved in patient care is essential to prevent medical errors and enhance patient experience and outcomes. [Level 5]