When a glucose result does not fit the patient, first determine whether it reflects patient physiology, the collection process, or an analytic limitation. Cells continue to consume glucose after collection, test methods respond to different chemical reactions, and hemoglobin A1c (HbA1c) depends on erythrocyte survival as well as average glycemia. This lesson covers each of those factors.
Carbohydrate structure and metabolism
Carbohydrates include monosaccharides, disaccharides, and larger polymers. Monosaccharides are polyhydroxy aldehydes or ketones and are classified by carbon number, carbonyl position, and stereochemistry. Glucose, galactose, and fructose are hexose monosaccharides. Aldoses have a terminal aldehyde in the open-chain form. Ketoses have an internal ketone. Maltose, lactose, and sucrose are disaccharides. Starch and glycogen are glucose polymers.
A free anomeric carbon allows a sugar to act as a reducing agent. Glucose, galactose, fructose, maltose, and lactose are reducing sugars. Both anomeric carbons in sucrose participate in its glycosidic bond. This chemistry explains why historical copper-reduction tests responded to several sugars.
After glucose enters cells, hexokinase in most tissues and glucokinase in hepatocytes and pancreatic beta cells convert it to glucose-6-phosphate (G6P). G6P then enters pathways that supply ATP, build glycogen, or support biosynthesis.1
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| Route | Main products and laboratory significance |
|---|---|
| Glycolysis | Glycolysis produces two pyruvate molecules and a net yield of two ATP in the cytosol. Erythrocytes depend on glycolysis because they lack mitochondria. Anaerobic metabolism converts pyruvate to lactate. |
| Tricarboxylic acid cycle | In aerobic metabolism, pyruvate is converted to acetyl coenzyme A, which enters the mitochondrial tricarboxylic acid cycle for energy production. |
| Pentose phosphate pathway | The pathway supplies erythrocytes with NADPH to maintain reduced glutathione. Its ribose-5-phosphate supports nucleotide synthesis. |
| Glycogenesis | Liver and muscle store glucose as glycogen after G6P is converted through glucose-1-phosphate and UDP-glucose. In the liver, glucose-6-phosphatase permits release of free glucose into blood. In muscle, glycogen supplies local energy. |
| Gluconeogenesis | Liver and kidney form glucose from lactate, glycerol, and glucogenic amino acids during fasting. |
Insulin promotes glucose uptake in muscle and adipose tissue and favors glycogen synthesis, glycolysis, and lipid synthesis. Glucagon promotes hepatic glycogen breakdown and gluconeogenesis. Epinephrine, cortisol, and growth hormone also raise circulating glucose during stress or fasting. Because pancreatic beta cells release insulin and C-peptide in equimolar amounts when proinsulin is cleaved, C-peptide is useful for assessing endogenous insulin secretion.
Glucose specimens and methods
Venous plasma is the recommended specimen for diabetes diagnosis. Plasma contains about 11% more water per liter than whole blood at a normal hematocrit, so its glucose concentration is about 11% higher. Glucose meters are therefore calibrated to report plasma-equivalent concentrations even when the sample is capillary whole blood.2
Cerebrospinal fluid (CSF) glucose follows plasma glucose with a delay and is often 60% to 70% of the paired plasma concentration. Collect a blood glucose specimen at the same time and interpret the CSF value against the laboratory's age- and method-specific interval. A low CSF-to-plasma ratio supports impaired glucose transport or glucose consumption within the central nervous system. Bacterial, fungal, and tuberculous meningitis can produce this pattern. Viral meningitis usually preserves the ratio. Prompt transport and processing limit continued glucose consumption by cells and microorganisms.1
Blood cells continue glycolysis after collection, and high leukocyte or erythrocyte counts increase the rate of glucose loss. Prompt separation of plasma from cells limits this loss. When immediate separation is unavailable, place the tube in an ice-water slurry and separate plasma within 30 minutes, or use a validated tube containing a glycolysis inhibitor that acts promptly. Sodium fluoride does not promptly prevent glucose loss, so clinically important glucose loss can occur during the first hours after collection. Tubes containing citrate buffer, sodium fluoride, and sodium EDTA (CFE) acidify the sample and suppress glycolysis promptly.2 Treat an unexpectedly low glucose as a possible handling artifact until the tube type, collection time, and separation time have been checked.
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| Method | Reaction | Main use and limitation |
|---|---|---|
| Copper reduction | A reducing substance converts cupric ion to cuprous ion and produces a colored precipitate | Historically used to screen urine for reducing substances. Drugs and nonglucose sugars can produce a positive reaction. |
| Glucose oxidase with peroxidase | Glucose oxidase forms hydrogen peroxide; peroxidase couples peroxide to a chromogen | Measures glucose selectively, but ascorbate, urate, bilirubin, hemoglobin, and other reducing substances may consume peroxide or alter the color, depending on the method. |
| Electrochemical glucose oxidase method | The sensor measures oxygen consumption or electron transfer during glucose oxidation | Common in blood glucose meters and some laboratory systems. Oxygen dependence and electroactive substances can affect results, depending on sensor design. |
| Hexokinase | Hexokinase forms G6P; G6P dehydrogenase forms NADPH measured at 340 nm | Standard laboratory method for plasma glucose. Limits for hemolysis, icterus, lipemia, and drug effects are analyzer-specific. |
For a fasting plasma glucose measurement, the patient must avoid caloric intake for at least 8 hours before collection. Preparation for a 75-g oral glucose tolerance test (OGTT) includes at least 150 g of carbohydrate daily for the 3 days preceding the test and a fast of at least 8 hours before collection. Outside pregnancy, laboratories measure fasting and 2-hour plasma glucose. For children, the glucose dose is 1.75 g/kg, up to 75 g. Vomiting, acute illness, altered gastrointestinal absorption, and drugs that change glucose metabolism can make the test result unreliable.3
Diabetes and prediabetes criteria
Diabetes mellitus is classified by cause. Type 1 diabetes results from beta-cell destruction and absolute insulin deficiency; islet-cell, insulin, glutamic acid decarboxylase, insulinoma-associated antigen 2, and zinc transporter 8 autoantibodies support autoimmune disease. An insulin autoantibody result requires the insulin-treatment history because exogenous insulin commonly produces insulin antibodies. Type 2 diabetes combines insulin resistance with progressive beta-cell dysfunction. Other specific forms include monogenic diabetes, pancreatic disease, endocrinopathies, and drug-associated diabetes. Gestational diabetes mellitus is diagnosed during the second or third trimester when overt diabetes was not clearly present before pregnancy.2,3
The 2026 American Diabetes Association (ADA) criteria define diabetes using laboratory plasma glucose or HbA1c. When unequivocal hyperglycemia is absent, two abnormal results confirm the diagnosis. The second result may come from repeating the same test promptly or from a different diagnostic test. One random plasma glucose result of at least 200 mg/dL, together with classic hyperglycemic symptoms or a hyperglycemic crisis, is sufficient to diagnose diabetes.3
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| Category | HbA1c | Fasting plasma glucose | 2-hour plasma glucose after 75-g OGTT | Random plasma glucose |
|---|---|---|---|---|
| Prediabetes | 5.7–6.4% (39–47 mmol/mol) | 100–125 mg/dL (5.6–6.9 mmol/L) | 140–199 mg/dL (7.8–11.0 mmol/L) | Not used to diagnose prediabetes |
| Diabetes | ≥6.5% (≥48 mmol/mol) | ≥126 mg/dL (≥7.0 mmol/L) | ≥200 mg/dL (≥11.1 mmol/L) | ≥200 mg/dL (≥11.1 mmol/L) with classic hyperglycemic symptoms or a hyperglycemic crisis |
Diagnostic limits are thresholds for a clinical decision, and they serve a different purpose from reference intervals. The performing laboratory validates any reference interval shown on a patient report.
The shape of the glucose response explains why the 2-hour value is the diagnostic measurement. After the 75-g load, a normal plasma glucose peaks within about 30 to 60 minutes and returns toward the fasting concentration by 2 hours because insulin is released promptly. With impaired glucose tolerance, the glucose peak is higher and later, the insulin response is delayed and often larger, and the 2-hour value stays between 140 and 199 mg/dL. In type 2 diabetes, the early insulin response is blunted and delayed, so glucose rises higher, peaks later, and remains at 200 mg/dL or above at 2 hours. Insulin concentrations vary widely between people, so the insulin response is described by its shape and timing.1
Gestational diabetes testing
At 24 to 28 weeks of gestation, ADA 2026 recognizes both the one-step strategy of the International Association of Diabetes and Pregnancy Study Groups and the two-step strategy. The American College of Obstetricians and Gynecologists supports the two-step strategy, which is also used in U.S. laboratories. The laboratory report must match the glucose load, collection times, and decision limits of the protocol used. These thresholds apply at 24 to 28 weeks of gestation. Early testing for overt diabetes uses the standard diagnostic criteria.3
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| Strategy | Collection and decision limits | Diagnostic rule |
|---|---|---|
| One-step | 75-g OGTT after an overnight fast of at least 8 hours: fasting 92 mg/dL, 1 hour 180 mg/dL, 2 hours 153 mg/dL | One value at or above its limit diagnoses gestational diabetes. |
| Two-step screen | Nonfasting 50-g glucose load with a 1-hour result; the local threshold is 130, 135, or 140 mg/dL | A positive screen is followed by the 100-g OGTT. |
| Two-step diagnostic test | 100-g OGTT while fasting: fasting 95 mg/dL, 1 hour 180 mg/dL, 2 hours 155 mg/dL, 3 hours 140 mg/dL | The Carpenter-Coustan criteria require two values at or above their limits; ACOG notes that one elevated value may be used. |
HbA1c and other glycated proteins
HbA1c forms through nonenzymatic attachment of glucose to the N-terminal valine of the hemoglobin A beta chain. The result reflects glycemia over roughly the preceding 2 to 3 months. Recent weeks contribute more strongly because circulating erythrocytes vary in age. Diagnostic testing requires a National Glycohemoglobin Standardization Program (NGSP)-certified method traceable to the Diabetes Control and Complications Trial reference assay. Point-of-care HbA1c diagnosis requires an FDA-approved diagnostic device used by trained personnel in a laboratory certified under the Clinical Laboratory Improvement Amendments (CLIA) that performs moderate- or high-complexity testing.3
Routine methods separate glycated from nonglycated hemoglobin by charge or structure.
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| Method group | Separation principle | Main interpretive issue |
|---|---|---|
| Ion-exchange chromatography or electrophoretic methods | Charge difference between hemoglobin fractions | Hemoglobin variants, fetal hemoglobin, temperature, and coelution can affect the reported HbA1c. The specific effect depends on the analyzer. |
| Immunoassay | Antibody recognition of the glycated N-terminal beta-chain epitope | Variant and fetal-hemoglobin effects depend on the recognized epitope and assay design. |
| Boronate affinity | Binding of cis-diol groups on glycated hemoglobin | Structural variants often have less direct analytic effect, but altered erythrocyte survival still changes interpretation. |
| Enzymatic assay | Proteolysis followed by measurement of glycated N-terminal beta-chain peptides | Assay design affects variant interference and total-hemoglobin measurement. |
The International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) reference system measures glycated and nonglycated N-terminal beta-chain hexapeptides by mass spectrometry or capillary electrophoresis. The master equation links routine results to this reference system:4
The estimated average glucose (eAG) equation converts HbA1c to glucose units:5
Estimated average glucose. Suppose the HbA1c is 7.8%.
eAG = 28.7 × 7.8 − 46.7 = 177 mg/dL
The eAG is a regression estimate, so measured glucose values vary around it.
HbA1c interpretation depends on erythrocyte age. Hemolysis, recent blood loss, erythropoietin use, and other causes of shortened survival produce an HbA1c result lower than the glycemic exposure would predict. Iron deficiency may raise HbA1c. Transfusion and pregnancy can alter the HbA1c-glycemia relationship. Hemoglobin variants may cause analytic interference, and the direction of the effect varies by method. When HbA1c and glucose results differ substantially, review the blood count, transfusion history, hemoglobin pattern, erythrocyte turnover, and the analyzer's interference table.6
When HbA is absent, as in HbSS, HbCC, HbEE, and HbSC, HbA1c cannot be measured, so do not report it. Glucose results or validated nonhemoglobin glycated-protein markers, such as fructosamine or glycated albumin, provide alternatives, each with its own limits.2
Fructosamine measures total glycated serum protein. Glycated albumin is reported relative to albumin. Both reflect a shorter interval of about 15 to 30 days and may help when erythrocyte turnover or a hemoglobin variant makes HbA1c difficult to interpret. Protein loss, altered albumin synthesis, and abnormal albumin turnover can change fructosamine and glycated albumin results. Interpret both tests with method-specific intervals. Standardization and outcome-based decision limits remain limited.2
Ketones and hyperglycemic crises
The liver forms acetoacetate, beta-hydroxybutyrate, and acetone when fatty-acid oxidation increases. Beta-hydroxybutyrate becomes the predominant ketoacid in diabetic ketoacidosis (DKA). The nitroprusside reaction produces a purple color with acetoacetate. Formulations containing glycine also react weakly with acetone. The nitroprusside reaction does not detect beta-hydroxybutyrate. Enzymatic methods using beta-hydroxybutyrate dehydrogenase quantify beta-hydroxybutyrate in blood.2
Direct measurement of beta-hydroxybutyrate in blood is preferred for DKA diagnosis and serial assessment, because nitroprusside does not detect the predominant ketoacid. Urine nitroprusside results may be modest early in DKA and then increase as beta-hydroxybutyrate converts to acetoacetate during recovery. Captopril and valproate can also cause false-positive nitroprusside results. Test serum or urine promptly, following the method-specific specimen instructions.7
Under the 2024 international consensus for DKA and hyperosmolar hyperglycemic state (HHS), all criteria in the relevant column must be met. Mixed presentations of DKA and HHS are frequent.7
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| Criterion | DKA | HHS |
|---|---|---|
| Glucose concentration or diabetes history | Plasma glucose ≥200 mg/dL (11.1 mmol/L) or a prior diagnosis of diabetes, regardless of presenting glucose | Plasma glucose ≥600 mg/dL (33.3 mmol/L) |
| Ketones | Beta-hydroxybutyrate ≥3.0 mmol/L or urine ketones ≥2+ | Beta-hydroxybutyrate <3.0 mmol/L or urine ketones <2+ |
| Acid-base status | pH <7.30 and/or bicarbonate <18 mmol/L | pH ≥7.30 and bicarbonate ≥15 mmol/L |
| Osmolality | Assessed separately from DKA criteria | Effective serum osmolality >300 mOsm/kg or total serum osmolality >320 mOsm/kg |
When beta-hydroxybutyrate is unavailable, the anion gap may support evaluation. Mixed acid-base disorders and hyperchloremic acidosis limit the anion gap's usefulness as a primary criterion for diagnosis or resolution.
Hypoglycemia evaluation
For people without diabetes, the Endocrine Society guideline evaluates a suspected hypoglycemic disorder only after Whipple's triad is documented: compatible symptoms or signs, a low plasma glucose measured by a reliable laboratory method, and resolution after glucose rises. Diagnosis requires all three elements and a laboratory-measured glucose value.8
For people with diabetes, ADA 2026 classifies hypoglycemia according to glucose level and the need for assistance.9
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| Level | Criterion |
|---|---|
| 1 | Glucose <70 mg/dL (3.9 mmol/L) and ≥54 mg/dL (3.0 mmol/L) |
| 2 | Glucose <54 mg/dL (3.0 mmol/L) |
| 3 | A severe event with altered mental and/or physical status that requires assistance to treat hypoglycemia, irrespective of glucose level |
When endogenous hyperinsulinism is suspected, collect the critical sample before glucose is given when feasible, because glucose changes the interpretive pattern. Emergency care takes priority over specimen collection. The sample may come from a spontaneous episode or a fast supervised by the clinical service. During symptoms or signs at a plasma glucose below 55 mg/dL, endogenous hyperinsulinism is documented when insulin is at least 3 µU/mL, C-peptide is at least 0.2 nmol/L, and proinsulin is at least 5 pmol/L, with a negative screen for insulin secretagogues. These measurements do not identify insulinoma by themselves. The clinical service is responsible for the fasting protocol and patient management.
A beta-hydroxybutyrate level no greater than 2.7 mmol/L plus a plasma-glucose rise of at least 25 mg/dL after intravenous glucagon supports insulin- or insulin-like growth factor-mediated hypoglycemia. Insulin antibodies help distinguish causes of hypoglycemia.8
Inherited carbohydrate disorders
Inherited defects can produce characteristic biochemical patterns. Molecular testing establishes most diagnoses. Enzyme analysis can provide additional evidence when a disorder-specific activity assay is available.
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| Disorder | Defect | Laboratory pattern and confirmation |
|---|---|---|
| Glycogen storage disease type I (GSD I) | GSD Ia: G6PC1 and deficient glucose-6-phosphatase activity; GSD Ib: SLC37A4 and impaired G6P transport | The laboratory pattern includes fasting hypoglycemia, lactic acidosis, hyperuricemia, hypertriglyceridemia, and hepatomegaly. Biallelic pathogenic or likely pathogenic variants establish the diagnosis. Liver enzyme testing may resolve selected GSD Ia cases.10 |
| Classic galactosemia | Severe galactose-1-phosphate uridylyltransferase deficiency from GALT variants | Newborn screening uses total galactose and/or erythrocyte GALT activity. Confirmation is supported by elevated erythrocyte galactose-1-phosphate, reduced GALT activity, and biallelic pathogenic or likely pathogenic GALT variants.11 |
| Hereditary fructose intolerance | Aldolase B deficiency from ALDOB variants | Exposure to fructose, sucrose, or sorbitol may produce hypoglycemia, lactic acidemia, hypophosphatemia, hyperuricemia, and liver or kidney injury. Biallelic pathogenic or likely pathogenic ALDOB variants provide first-line confirmation. A diagnostic fructose challenge can cause severe hypoglycemia and must be avoided.12 |
Deficiencies of hepatic glycogen synthase, fructose-1,6-bisphosphatase, phosphoenolpyruvate carboxykinase, and pyruvate carboxylase can also produce fasting hypoglycemia. Biochemical and molecular testing distinguishes among these disorders.1
Practice
References
- Rifai N, Chiu RWK, Young I, Burnham CAD, Wittwer CT, eds. Tietz Textbook of Laboratory Medicine. 7th ed. Elsevier; 2023. Back to text
- Sacks DB, Arnold M, Bakris GL, et al. Guidelines and recommendations for laboratory analysis in the diagnosis and management of diabetes mellitus. Diabetes Care. 2023;46(10):e151-e199. doi:10.2337/dci23-0036 Back to text
- American Diabetes Association Professional Practice Committee for Diabetes. 2. Diagnosis and classification of diabetes: Standards of Care in Diabetes-2026. Diabetes Care. 2026;49(suppl 1):S27-S49. doi:10.2337/dc26-S002 Back to text
- National Glycohemoglobin Standardization Program. IFCC standardization of HbA1c. Accessed August 28, 2026. https://ngsp.org/ifcc.asp Back to text
- National Glycohemoglobin Standardization Program. HbA1c and estimated average glucose. Accessed August 28, 2026. https://ngsp.org/A1ceAG.asp Back to text
- National Glycohemoglobin Standardization Program. Factors that interfere with HbA1c test results. Updated June 23, 2026. Accessed August 28, 2026. https://ngsp.org/factors.asp Back to text
- Umpierrez GE, Davis GM, ElSayed NA, et al. Hyperglycemic crises in adults with diabetes: a consensus report. Diabetes Care. 2024;47(8):1257-1275. doi:10.2337/dci24-0032 Back to text
- Cryer PE, Axelrod L, Grossman AB, et al. Evaluation and management of adult hypoglycemic disorders: an Endocrine Society clinical practice guideline. J Clin Endocrinol Metab. 2009;94(3):709-728. doi:10.1210/jc.2008-1410 Back to text
- American Diabetes Association Professional Practice Committee for Diabetes. 6. Glycemic goals, hypoglycemia, and hyperglycemic crises: Standards of Care in Diabetes-2026. Diabetes Care. 2026;49(suppl 1):S132-S149. doi:10.2337/dc26-S006 Back to text
- Bali DS, El-Gharbawy A, Austin S, Pendyal S, Kishnani PS. Glycogen storage disease type I. Updated October 14, 2021. In: Adam MP, Bick S, Mirzaa GM, et al, eds. GeneReviews. University of Washington, Seattle; 1993-2026. Accessed August 28, 2026. NCBI Bookshelf Back to text
- Berry GT. Classic galactosemia and clinical variant galactosemia. GeneReviews. Updated March 11, 2021. Accessed August 28, 2026. NCBI Bookshelf Back to text
- Gaughan S, Ayres L, Baker PR II. Hereditary fructose intolerance. GeneReviews. Updated February 18, 2021. Accessed August 28, 2026. NCBI Bookshelf Back to text