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Laboratory Diagnosis

Laboratory Findings


a) Glucosuria

Glycosuria allows for a good first-line screening test for diabetes mellitus. Normally glucose does not appear in urine until the plasma glucose rises above 10 mmol/L or more. But in certain individuals due to low renal threshold glucose may be present despite normal blood glucose levels. Conversely renal threshold increases with age so many diabetics may not have Glycosuria despite high blood sugar levels.

A specific and convenient method to detect glucosuria is the paper strip impregnated with glucose oxidase and a chromogen system (Clinistix, Diastix), which is sensitive to as little as 0.1% glucose in urine. Diastix can be directly applied to the urinary stream, and differing color responses of the indicator strip reflect glucose concentration.

A normal renal threshold for glucose as well as reliable bladder emptying is essential for interpretation.


Qualitative detection of ketone bodies can be accomplished by nitroprusside tests (Acetest or Ketostix). Although these tests do not detect Beta-hydroxybutyric acid, which lacks a ketone group, the semiquantitative estimation of ketonuria thus obtained is nonetheless usually adequate for clinical purposes. Ketone bodies may be present in normal subject as a result of simple prolonged fasting.

c) Microalbuminuria

Microalbuminuria may be defined as an albumin excretion rate intermediate between normality (2.5-25 mg/day) and macroalbuminuria (250mg/day). The small increase in urinary albumin excretion is not detected by simple albumin stick tests and requires confirmation by careful quantization in a 24 hr. urine specimen. The importance of micro- albuminuria in the diabetic patient is that it is a signal of early reversible renal damage. Performing an albumin-to-creatinine ratio is probably easiest.

Unlike type 1 diabetes mellitus, in which microalbuminuria is a good indicator of early kidney damage, microalbuminuria is a common finding (even at diagnosis) in type 2 diabetes mellitus and is a risk factor for macro vascular (especially coronary heart) disease. It is a weaker predictor for future kidney disease in type 2 diabetes mellitus.

2) Blood Testing Procedures

a) Glucose tolerance test

Methodology and normal fasting glucose

Plasma or serum from venous blood samples has the advantage over whole blood of providing values for glucose that are independent of Haemtocrit and that reflect the glucose concentration to which body tissues are exposed. For these reasons, and because plasma and serum are more readily measured on automated equipment, they are used in most laboratories. If serum is used or if plasma is collected from tubes that lack an agent to block glucose metabolism (such as fluoride), samples should be refrigerated and separated within 1 hour after collection. The glucose concentration is 10–15% higher in plasma or serum than in whole blood because structural components of blood cells are absent.

Fasting blood Glucose

Fasting blood glucose is measured after an overnight fast of 10 hrs. Fasting blood glucose estimation is better than random blood glucose. FPG < 5.6 mmol/L (100 mg/dL) is considered normal; (2) FPG = 5.6–6.9 mmol/L (100–125 mg/dL) is defined as IFG; and (3) FPG >7.0 mmol/L (126 mg/dL) warrants the diagnosis of DM.

Criteria for laboratory confirmation of diabetes mellitus

If the fasting plasma glucose level is 126 mg/dL or higher on more than one occasion, further evaluation of the patient with a glucose challenge is unnecessary. However, when fasting plasma glucose is less than 126 mg/dL in suspected cases, a standardized oral glucose tolerance test may be done .

75 g of glucose dissolved in 300 mL of water is given after an overnight fast to a person who has been receiving at least 150–200 g of carbohydrate daily for 3 days before the test. The data is interpreted as follows-

The Diabetes Expert Committee criteria for evaluating the standard oral glucose tolerance test.

For proper evaluation of the test, the subjects should be normally active and free from acute illness. Medications that may impair glucose tolerance include diuretics, contraceptive drugs, glucocorticoids, niacin, and phenytoin should be avoided on that day.

Random blood Glucose

Random is defined as without regard to time since the last meal.

RBG measurement is required only during emergency. The current criteria for the diagnosis of DM emphasize that the FPG is the most reliable and convenient test for identifying DM in asymptomatic individuals. A random plasma glucose concentration >11.1 mmol/L (200 mg/dL) accompanied by classic symptoms of DM (polyuria, polydipsia, weight loss) is sufficient for the diagnosis of DM

b) Glycated hemoglobin (Hb1C) measurements

Hemoglobin becomes glycated by ketoamine reactions between glucose and other sugars and the free amino groups on the alpha and beta chains. Only glycation of the N-terminal valine of the beta chain imparts sufficient negative charge to the hemoglobin molecule to allow separation by charge dependent techniques. These charge separated hemoglobin are collectively referred to as hemoglobin A1 (HbA1). The major form of HbA1 is hemoglobin A1c (HbA1c) where glucose is the carbohydrate. HbA1c comprises 4–6% of total hemoglobin A1. The remaining HbA1 species contain fructose-1, 6 bisphosphate (HbA1a1); glucose-6-phosphate (HbA1a2); and unknown carbohydrate moiety (HbA1b). The hemoglobin A1c fraction is abnormally elevated in diabetic persons with chronic hyperglycemia.

Methods for measuring HbA1c include electrophoresis, cation-exchange chromatography, boronate affinity chromatography, and immunoassays. Office-based immunoassays using capillary blood give a result in about 9 minutes and this allows for immediate feedback to the patients regarding their glycemic control.

Since glycohemoglobins circulate within red blood cells whose life span lasts up to 120 days, they generally reflect the state of glycemia over the preceding 8–12 weeks, thereby providing an improved method of assessing diabetic control. Measurements should be made in patients with either type of diabetes mellitus at 3- to 4-month intervals so that adjustments in therapy can be made.

In patients monitoring their own blood glucose levels, HbA1c values provide a valuable check on the accuracy of monitoring.

Use of HbA1c for screening is controversial. Sensitivity in detecting known diabetes cases by HbA1c measurements is only 85%, indicating that diabetes cannot be excluded by a normal value. On the other hand, elevated HbA1c assays are fairly specific (91%) in identifying the presence of diabetes.

The accuracy of HbA1c values can be affected by hemoglobin variants or derivatives; the effect depends on the specific hemoglobin variant or derivative and the specific assay used. Immunoassays that use an antibody to the glycated amino terminus of beta globin do not recognize the terminus of the gamma globin of hemoglobin F. Thus, in patients with high levels of hemoglobin F, immunoassays give falsely low values of HbA1c. Cation-exchange chromatography separates hemoglobin species by charge differences. Hemoglobin variants that co-elute with HbA1c can lead to an overestimation of the HbA1c value. Chemically modified derivatives of hemoglobin such as carbamoylation (in renal failure) or Acetylation (high-dose aspirin therapy) can similarly co-elute with HbA1c by some assay methods.

Any condition that shortens erythrocyte survival or decreases mean erythrocyte age (eg, recovery from acute blood loss, hemolytic anemia) will falsely lower HbA1c irrespective of the assay method used. Alternative methods such as fructosamine should be considered for these patients. Vitamins C and E are reported to falsely lower test results possibly by inhibiting glycation of hemoglobin.

c) Serum fructosamine

Serum fructosamine is formed by nonenzymatic glycosylation of serum proteins (predominantly albumin). Since serum albumin has a much shorter half-life than hemoglobin, serum fructosamine generally reflects the state of glycemic control for only the preceding 1–2 weeks. Reductions in serum albumin (eg, nephrotic state or hepatic disease) will lower the serum fructosamine value. When abnormal hemoglobins or hemolytic states affect the interpretation of glycohemoglobins or when a narrower time frame is required, such as for ascertaining glycemic control at the time of conception in a diabetic woman who has recently become pregnant, serum fructosamine assays offer some advantage. Normal values vary in relation to the serum albumin concentration and are 1.5–2.4 mmol/L when the serum albumin level is 5 g/dL.

d) Self-monitoring of blood glucose

Capillary blood glucose measurements performed by patients themselves, as outpatients, are extremely useful. In type 1 patients in whom “tight” metabolic control is attempted, they are indispensable. There are several paper strip (glucose oxidase, glucose dehydrogenase, or hexokinase) methods for measuring glucose on capillary blood samples. A reflectance photometer or an amperometric system is then used to measure the reaction that takes place on the reagent strip.

e) Lipid profile

Circulating lipoproteins are just as dependent on insulin as is the plasma glucose. In type 1 diabetes, moderately deficient control of hyperglycemia is associated with only a slight elevation of LDL cholesterol and serum triglycerides and little if any change in HDL cholesterol. Once the hyperglycemia is corrected, lipoprotein levels are generally normal. However, in obese patients with type 2 diabetes, a distinct “diabetic dyslipidemia” is characteristic of the insulin resistance syndrome. Its features are a high serum triglyceride level (300–400 mg/dL), a low HDL cholesterol (less than 30 mg/dL), and a qualitative change in LDL particles, producing a smaller dense particle whose membrane carries supranormal amounts of free cholesterol. These smaller dense LDL particles are more susceptible to oxidation, which renders them more atherogenic. Since a low HDL cholesterol is a major feature predisposing to macrovascular disease, the term “dyslipidemia” has preempted the term “hyperlipidemia,” which mainly denoted the elevated triglycerides. Measures designed to correct the obesity and hyperglycemia, such as exercise, diet, and hypoglycemic therapy, are the treatment of choice for diabetic dyslipidemia, and in occasional patients in whom normal weight was achieved, all features of the lipoprotein abnormalities cleared

f) Additional Tests

In addition to the standard laboratory evaluation, the patient should be screened for DM-associated conditions (e.g., kidney, liver and thyroid dysfunction). Individuals at high risk for cardiovascular disease should be screened for asymptomatic CAD by appropriate cardiac stress testing, when indicated.

The classification of the type of DM may be facilitated by laboratory assessments. Serum insulin or C-peptide measurements do not always distinguish type 1 from type 2 DM, but a low C-peptide level confirms a patient’s need for insulin. Many individuals with new-onset type 1 DM retain some C-peptide production.

 Insulin levels generally are high early in the course of type 2 diabetes mellitus and gradually wane over time. Stimulated C-peptide concentrations (after a standard meal challenge such as Sustacal or after glucagon) are somewhat preserved until late in the course of type 2 diabetes mellitus. Absence of a C-peptide response to carbohydrate ingestion may indicate total beta cell failure.

Measurement of islet cell antibodies at the time of diabetes onset may be useful if the type of DM is not clear based on the characteristics described above. Antibodies to insulin, islet cells, or Glutamic acid decarboxylase (GAD) are absent in type 2 diabetes mellitus.

Latent autoimmune diabetes of adults, or LADA, is a form of slow-onset type 1 diabetes that occurs in middle-aged (usually white) adults. It can be differentiated from type 2 diabetes by measuring anti-GAD65 antibodies. Such patients may respond to insulin secretagogue for a brief period (months).

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