For the determination of urea in serum, plasma and urine

(for in vitro diagnostic use only)

Summary

Urea is the end product of protein metabolism. It is

synthesized in the liver from the ammonia produced by

the catabolism of amino acids. It is transported by the

blood to the kidneys from where it is excreted. Increased

levels are found in renal diseases, urinary obstructions,

shock, congestive heart failure and burns. Decreased

levels are found in liver failure and pregnancy.

Principle

Urease hydrolyzes urea to ammonia and CO2. The ammonia

formed further reacts with a phenolic chromogen and

hypochlorite to form a green colored complex. Intensity of

the color formed is directly proportional to the amount of

urea present in the sample.

 Urease

Urea + H2O Ammonia + CO2

Ammonia + Phenolic chromogen + Hypochlorite

 Green colored complex

Normal Reference Values

Serum/plasma : 14–40 mg/dL

Urine : Upto 20 g/L

It is recommended that each laboratory establish its

own normal range representing its patient population.

Contents 75 assays 3 × 75 assays

L1: Buffer reagent 75 mL 3 × 75 mL

L2: Enzyme reagent 7.5 mL 3 × 7.5 mL

L3: Chromogen reagent 15 mL 45 mL

S: Urea standard (40 mg/dL) 5 mL 5 mL

Storage/Stability

Contents are stable at 2–8°C till the expiry mentioned on

the labels.

Reagent Preparation

Reagents are ready to use for the given procedure.

Working enzyme reagent: For the flexibility and

convenience in performing large assay series, a working

enzyme reagent may be made by pouring 1 bottle of L2

(Enzyme reagent) into 1 bottle of L1 (Buffer reagent). For

smaller series combine 10 parts of L1 (Buffer reagent) and

1 part of L2 (Enzyme reagent). Use 1 mL of the working

reagent per assay instead of 1 mL of L1 and 0.1 mL of L2

as given in the procedure. The working enzyme reagent is

stable for at least 4 weeks when stored at 2–8°C.

Working chromogen reagent: For larger volume cuvettes,

dilute 1 part of L3 (Chromogen reagent) with 4 parts of

fresh ammonia free distilled/deionised water. Use 1 ml

of working chromogen instead of 0.2 mL in the assay. The

working chromogen reagent is stable for atleast 8 weeks

when stored at 2–8°C in a tightly stoppered plastic bottle.

Sample Material

Serum, plasma, urine. Dilute urine 1 + 49 with distilled

water before the assay (results × 50). Urea is reported to be

stable in the serum for 5 days when stored 2–8°C.

Procedure

Wavelength/filter : 570 nm (Hg 578 nm)/yellow

Temperature : 37°C/RT

Light path : 1 cm

Pipette into clean dry test tubes labeled as blank (B),

standard (S), and test (T):

Addition

Sequence

B

(mL)

S

(mL)

T

(mL)

Buffer reagent (L1 ) 1.0 1.0 1.0

Enzyme reagent (L2) 0.1 0.1 0.1

Distilled water 0.01 — —

Urea standard (S) 0.01 —

Sample — — 0.01

Mix well and incubate for 5 minutes at 37°C or 10 minutes at RT

(25°C)

Chromogen reagent (L3) 0.2 0.2 0.2

470 Concise Book of Medical Laboratory Technology: Methods and Interpretations Mix well and incubate for 5 minutes at 37°C or

10 minutes at RT (25°C). Measure the absorbance of the

Standard (Abs S), and Test sample (Abs T) against the

Blank, within 60 minutes.

Calculations

 Abs T

Urea in mg/dL = ________ × 40 Abs S

Urea nitrogen in mg/dL = Urea in mg/dL × 0.467

Linearity

This procedure is linear upto 250 mg/dL. Using the working

chromogen reagent (1 mL) the linearity is increased to

400 mg/dL. If values exceed this limit, dilute the serum

with normal saline (NaCL 0.9%) and repeat the assay.

Calculate the value using the proper dilution factor.

Note

Any contamination by ammonia or ammonium salts lead

to erroneous results, hence plasma should not be collected

with fluoride or heparin ammonium salts. The working

enzyme reagent is not stable at elevated temperatures and

should be stored back at 2–8°C immediately after use. The

chromogen reagent contains chlorine. The bottle should

be opened only when required and closed tightly after use

to prevent the loss of active chlorine.

System Parameters

Reaction : End point No. of read :

Wavelength : 570 nm Interval :

Zero setting :  Reagent blank Sample

volume

: 0.01 mL

Incubation

temperature

: 37°C/RT Reagent

volume

: 1.30 mL

Incubated

time

:  5 min + 5 min

or

10 min +10 min

Standard

factor

React slope

: 40 mg/dL

: Increasing

Delay time : Linearity : 250 mg/dL

Read time : .... Units : mg/dL

Urea (GLDH Kinetic Method)

(Courtesy: Tulip Group of Companies)

For the determination of urea in serum or plasma (for

in vitro diagnostic use only).

Summary

Urea is the end product of protein metabolism. It is

synthesized in the liver from the ammonia produced by

the catabolism of amino acids. It is transported by the

blood to the kidneys from where it is excreted. Increased

levels are found in renal diseases, urinary obstructions,

shock, congestive heart failure and burns. Decreased

levels are found in liver failure and pregnancy.

Principle

Urease hydrolyzes urea to ammonia and CO2. The ammonia

formed further combines with a ketoglutarate and NADH

to form glutamate and NAD. The rate of oxidation of NADH

to NAD is measured as a decrease in absorbance in a fixed

time which is proportional to the urea concentration in the

sample.

 Urease

Urea + H2O + 2 H+ 2 NH4 + CO2

 GLDH

2 NH4

+ + 2 α Ketoglutarate + ↓

 2 NADH 2 L-glutamate + 2

 NAD+ + 2 H2O

Normal Reference Values

Serum/plasma : 14–40 mg/dL

Urine : Upto 20 g/L

It is recommended that each laboratory establish its

own normal range representing its patient population.

Contents 75 mL 2 ×75 mL

L1: Enzyme reagent 60 mL 2 × 60 mL

L2: Starter reagent 15 mL 2 × 15 mL

S: Urea standard (40 mg/dL) 5 mL 5 mL

Storage/stability

Contents are stable at 2–8°C till the expiry mentioned on

the labels.

Reagent Preparation

Reagents are ready to use.

Working reagent: For sample start assays a single reagent

is required. Pour the contents of 1 bottle of L2 (Starter

Reagent) into 1 bottle of L1 (Enzyme reagent).

This working reagent is stable for at least 10 days when

stored at 2–8°C. Alternatively for flexibility as much of

working reagent may be made as and when desired by

mixing together 4 parts of L1 (Enzyme reagent) and 1

part of L2 (Starter reagent). Alternatively 0.8 mL of L1 and

0.2 mL of L2 may also be used instead of 1 mL of the

working reagent directly during the assay.

Clinical Chemistry 471

Sample Material

Serum, plasma, urine. Dilute urine 1 + 49 with distilled

water before the assay (results × 50 ). Urea is reported to be

stable in the serum for 5 days when stored at 2–8°C.

Procedure

Wavelength/filter : 340 nm

Temperature : 37°C/30°C/25°C

Light path : 1 cm

Substrate Start Assay

Pipette into a clean dry test tube labeled standard (S) or

test (T):

Addition

Sequence

(S)/(T)

37°C/ 30°C/25°C

Enzyme reagent (L1) 0.8 mL

 

Fecal Urobilinogen

If the hepatobiliary system is functioning, fecal

urobilinogen varies directly with rate of red cell hemolysis.

Fecal urobilinogen increased occurs when blood destruction is increased and biliary obstruction is relieved.

In case of hemolysis, the daily excretion is related to

the existing total body hemoglobin mass. If there is a

reduced total body hemoglobin mass, accelerated rates

of hemolysis may only yield an amount of urobilinogen

that would be within normal limits for an individual with

normal hemoglobin mass.

Fecal urobilinogen absent occurs with exclusion of

bilirubin from the gut in complete biliary tract obstruction

and in extreme cases of hepatocellular disease. Absence

of urobilinogen in feces is important in indicating biliary

tract obstruction, persistent absence is a strong indication

of malignant obstructive disease. Decreased fecal

urobilinogen excretion may occur when antibiotics which

alter intestinal flora are used (tetracyclines, streptomycin,

etc.).

Bromsulphalein (Sulfobromophthalein) Excretion

Test

Sulfobromophthalein sodium (Bromsulphalein, BSP, is

a dye which is bound avidly by albumin, the complex is

picked up by the liver cell, and the BSP is transported into

the microsomes, conjugated and excreted in the bile, in a

manner analogous to bilirubin.

BSP is given intravenously and its amount present in the

blood after 30, 45 or 60 minutes indicate hepatic function.

The greater the liver function loss, greater the amount

BSP present in blood (this test is of no significant value in

differential diagnosis). BSP retention in blood conjugated/

unconjugated runs parallel to bilirubin related disorders.

In the presence of fevers, administration of anabolic

steroids, Tele-paque, Dubin-Johnson syndrome and in

Gilbert’s disease, BSP retention is increased. In the latter

two disorders the uptake by liver cells is normal (so normal

retention at 45 minutes) but after getting conjugated it

regurgitates back into blood (so BSP retention is marked at

3 hours period).

Method

1. Lipemic/jaundiced sera may cause interference. In any

case one should not perform the test in the presence

of acute liver/gallbladder disease.

2. Know the patient’s weight, inject 5 mg BSP/kg body

weight intravenously, over a period of 60 seconds.

Having injected set the time to 45 minutes.

3. After 45 minutes withdraw 8–10 mL blood from the

other arm, let it clot, remove serum after centrifuging.

4. Take 2 test tubes marked test and blank:

Test Blank

1.0 mL serum

+4.0 mL N/10 NaOH mix by

gentle inversion

1.0 mL serum

+4.0 mL N/10 HCl mix by

gentle inversion

 The full reddish color develops in the alkaline solution,

and the dye is colorless in acid solution.

5. Read the absorbance of test at 575 nm (or 590 nm)

setting the zero with the blank.

6. Refer the absorbance reading to the calibration curve

to obtain the percentage of the dye dose remaining

in the blood after the 45 minutes interval. Report as

percentage of dye retention afterminutes,” giving

the dose used. Normal retention is up to 4% (average

2.8%).

Calibration: Into a 1 liter volumetric flask pipette very

accurately 2.0 mL of 5% BSP. Dilute to the mark with

distilled water and mix well. Into 4 clean test tubes pipette

accurately the following amounts of the diluted dye: 0.25,

0.50, 0.75, and 1.0 mL. Make each to 1.0 mL with distilled

water. To each tube add exactly 4.0 mL of N/10 NaOH.

Mix by inversion and read the absorbance at 575/590 nm.

These standards correspond to values of 25, 50, 75 and

100% retention in the test. Plotted on graph paper, the

readings should fall on a straight line passing through the

origin.

Conditions Associated with Increased BSP Retention

Hepatobiliary System

¾ Jaundice from any cause except Gilbert’s syndrome

¾ Viral hepatitis

¾ Toxic hepatitis

¾ Fatty liver

¾ Cirrhosis

¾ Bile duct obstruction

¾ Metastatic carcinoma

¾ Lymphomatous or leukemic infiltration

¾ Granulomatous inflammation

Liver Function Tests 457

¾ Amyloidosis

¾ Dubin-Johnson syndrome.

Extrahepatic Conditions

¾ Congestive heart failure

¾ Fever above 39°C

¾ Oral contraceptive use

¾ Prolonged fasting or malnutrition

¾ Contrast media used for gallbladder examination.

Artefacts

¾ Obesity

• Spuriously high retention because excessive weight

results in excessive dose

¾ Hypoalbuminemia

• Spuriously low retention because binding is reduced

¾ Ascites

• Spuriously low retention because the dye enters the

ascitic fluid

¾ Proteinuria

• Spuriously low retention because albumin bound dye

enters urine.

EVALUATION OF SYNTHESIS IN LIVER

Serum Proteins (Albumin Especially)

Since serum albumin and a small fraction of globulin

are synthesized in liver, serum proteins are affected both

quantitatively and qualitatively in liver disease. In any

disease causing hepatocellular damage, the concentration

of serum albumin decreases. In many liver disorders,

serum globulins may rise to such a level so as to maintain

normal or increased total protein concentration even

when there is severe albumin depletion.

The changing levels of serum albumin thus provide

valuable indices of severity, progress, and prognosis

in hepatic disease. Decreased albumin and elevated

globulins in serum indicate hepatocellular origin of

jaundice or liver disease. In obstructive jaundice, serum

protein changes occur late, after secondary hepatocellular

damage has occurred. Cholangitis and biliary cirrhosis,

however, result in liver damage which may not be accompanied by protein alteration. Furthermore, serum protein

changes may return to normal before convalescence from

hepatitis is complete. However, liver disease is not the only

cause of serum protein alterations.

Chemical methods and electrophoretic methods are

available for serum proteins estimation. Electrophoresis is

most precise and specific way of assessing serum proteins.

The flocculation and turbidity methods crudely estimate

globulins and hence are not specific and obsolete in

today’s context.

Prothrombin Concentration

Deficiency of prothrombin may occur as a result of:

1. Inadequate absorption of bile from the intestinal tract,

or

2. Inability of a damaged liver to convert vitamin K to

prothrombin.

A normal prothrombin concentration does not rule out

abnormal liver function.

Low Prothrombin in Presence of Jaundice

When a low prothrombin level is found in a jaundice

patient, give 2–4 mg vitamin K, IV or IM, and measure

prothrombin concentration later.

1. Return to normalcy of prothrombin concentration

(85–100% of normal) indicates that the capacity of liver

cells to synthesize prothrombin is good.

2. A poor response implies hepatocellular disease, either

primary or following prolonged obstructive disease.

Low Prothrombin in the Absence of Jaundice

In the absence of jaundice, a low prothrombin level usually

indicates serious liver damage, and no response to large

doses (60–70 mg) of parenteral water-soluble vitamin K

confirms it. This is true if jaundice is also present.

Cholesterol and its Esters

Decrease of Both Substances

Associated with extensive destruction of liver parenchyma

is reduction in serum levels of cholesterol and cholesterol

esters, extremely low concentration implies a poor

prognosis. Persistently low cholesterol ester concentration

or ester/total cholesterol ratio indicates continuing

hepatocellular damage, a rise in cholesterol ester is

considered as a good sign and heralds improvement.

Increase of Total but Decrease of Esters

Accompanying biliary obstruction is usually a rise in total

cholesterol, but the cholesterol ester concentration is

often unaffected. The determination of cholesterol ester,

however, is not a fruitful exercise clinically.

Detoxification

The liver removes noxious materials or renders them

harmless by conjugation of toxic substances with amino

458 Concise Book of Medical Laboratory Technology: Methods and Interpretations acids, glucuronate and inorganic radicals (e.g. sulfate), by

oxidation or reduction, by excretion, etc.

Hippuric Acid Test

This test depends upon conjugation by liver of sodium

benzoate with glycine to produce hippuric acid, which

is excreted in the urine. It is preferrable to give sodium

benzoate-1.77 g-IV (instead of orally in which case the

absorption may be irregular), one hour later at least

0.7 g of hippuric acid should be excreted in the urine.

Consideration of low values is permissible only if impaired

renal function is ruled out for retention of hippuric acid.

EVALUATION OF ENZYME ACTIVITY

Serum Transaminases

Liver and muscles are rich in enzymes of Kreb’s cycle.

Among such enzymes is a group responsible for transfer of

NH2 groups from amino acids to keto acids, thus, providing

for metabolism of amino acids. Destruction of muscle or

of liver cells releases the enzymes, with consequent rise in

their values in plasma. In obstructive jaundice and more

so in acute hepatitis, the serum levels of SGOT and SGPT

rise to very high levels (300-1500 units, normal being 5-40

units), as does LDH concentration (normal concentration,

200-450 units). Chronic hepatitis may produce

moderate elevations of serum transaminases. Liver cell

destruction incident to neoplastic disease metastatic to

the liver produces moderate elevation of transaminases

concentration in the serum.

In many cases, there seems to be a correlation between

the differences in the degree of elevation of SGOT and

SGPT and the cause of jaundice. Rise of SGPT is greater

than elevation of SGOT in extrahepatic obstruction, acute

hepatitis and toxic hepatitis, the reverse is true in cirrhosis

of liver, intrahepatic neoplasm, and hemolytic jaundice.

Serum Alkaline Phosphatase

The concentration of this enzyme often increases in the

plasma of an icteric patient. It is normally present in the

liver and excreted in the bile so that elevation of serum

alkaline phosphatase may be a manifestation of retention;

this is a convenient explanation for the observation that

serum alkaline phosphatase concentration increases in

obstructive jaundice. In acute and chronic hepatocellular

disease, serum alkaline phosphatase is raised, but not to

the extent typical of obstructive jaundice. In hemolytic

jaundice, normal levels are the rule. In some cases of metastatic carcinoma of liver, serum alkaline phosphatase may

rise in the absence of jaundice. It should be kept in mind

that phosphatase levels may be normal early in obstructive

disease and with relief of obstruction. Pregnancy and such

diseases as Paget’s disease of bone, hyperparathyroidism,

and rickets/osteomalacia, are also associated with

elevated serum alkaline phosphatase concentration and

these must be ruled out.

SUGGESTED LIVER FUNCTION TESTS

A. Jaundice Absent

Urine bilirubin, urine urobilinogen, serum bilirubin, BSP

excretion, transaminases.

B. Jaundice Present

As mentioned above (except BSP excretion), plus alkaline

phosphatase,

 

Diabetes Mellitus:

Laboratory Diagnosis

C H A P T E R

DIABETES MELLITUS

Diabetes mellitus is a chronic metabolic disorder with

vascular components that is characterized by disturbances

in carbohydrate, lipid and protein metabolism. So,

hyperglycemia and glycosuria reflect the major metabolic

lesion in carbohydrate metabolism, with secondary

metabolic disturbances in proteins (gluconeogenesis) and

lipids (ketosis and hypercholesterolemia).

With hyperglycemia, renal glycosuria occurs with

an osmotic diuresis (polyuria) ultimately leads to

dehydration and associated polydipsia (increased thirst).

Glycogenolysis and gluconeogenesis (protein depletion)

are augmented to generate glucose that contributes to or

sustains hyperglycemia. Muscle glycogen cannot contribute glucose directly to the blood because of the absence

of glucose-6-phosphatase. A failure of glucose to penetrate

adipose tissue cells mobilizes fat and produces a rise in

the free fatty acid and triglycerides in the liver. A diabetic

fatty liver may result from the absence of lipoprotein

synthesis when protein synthesis is compromised by

accelerated gluconeogenesis (negative nitrogen balance).

When glucose oxidation is impaired, fatty acids form the

major source of energy and generate an excess of acetyl

coenzyme A that cannot be oxidized to water and carbon

dioxide or be disposed of in other metabolic routes. The

condensation of two carbon fragments of acetyl coenzyme

A results in formation of ketone bodies, ketonemia

and ketonuria. The three ketone bodies are acetone, β

hydroxybutyric acid and acetoacetic acid. Ketoacidosis is

the hallmark of potentially fatal complications of diabetes

mellitus.

Classification and Causes of Diabetes

Primary

¾ Maturity-onset (adult) type

¾ Growth-onset (juvenile) type

¾ Hyperpituitarism

• Pituitary basophilism

• Acromegaly.

¾ Hyperadrenalism

• Cortical: Cushing’s syndrome, aldosteronism

• Medullary: Pheochromocytoma.

¾ Hyperthyroidism

¾ Iatrogenic

• Corticosteroids and ACTH

• Growth hormone

• Thyroid extract and triiodothyronine.

Destruction of Pancreatic Islets

¾ Surgical removal of pancreas

¾ Hemochromatosis

¾ Fibrocystic disease of pancreas

¾ Neoplasm.

Miscellaneous

¾ Diuretics and derivatives (thiazide therapy)

¾ Stress reactions, surgery and pregnancy

¾ Starvation and low carbohydrate intake.

The course of the disease can be divided into four

stages:

1. Prediabetes

2. Suspected diabetes

3. Chemical or latent diabetes

4. Overt diabetes.

Diabetes Mellitus: Laboratory Diagnosis 435

The period from birth until the first evidence of the

disease characterizes prediabetes. In suspected diabetes,

the patient displays an abnormal glucose tolerance test

(GTT) or even diabetic symptoms after stressful influences

(e.g. obesity, pregnancy, trauma and infections), but

usually is normal in all respects. In chemical or latent

diabetes, there are no signs or symptoms of disease but

an abnormal GTT or fasting hyperglycemia are evident

when the patient is not under stress. With overt diabetes,

symptoms of polyuria, polydipsia and weight loss (and

possibly ketoacidosis) are often associated with fasting

hyperglycemia and glycosuria. For diagnosis of diabetes

in individuals with marked glucose intolerance, the

provocative tests should not be performed (as in an

insulin requiring diabetic). In patients who have neither

glycosuria nor fasting hyperglycemia—in these individuals

provocative tests may be needed to demonstrate impaired

glucose tolerance. Glycosuria associated with ketonuria is

almost always pathognomonic of diabetes mellitus.

Screening Tests

These include urine and blood glucose estimations:

Urine Glucose (Methods Mentioned Elsewhere)

While evaluating glycosuria, it should be remembered that

venous “true glucose” must exceed 180 mg% of blood before

any glucose will spill over into urine (renal threshold). In

diabetic nephropathy, the renal threshold may be elevated

considerably (very little filtration apparatus left, i.e.

glomeruli) even in the presence of hyperglycemia. Also,

the renal threshold increases with age, and in some elderly

patients no glycosuria will be present with serum levels of

200 mg% of glucose.

Fasting Blood Sugar

For this, plasma is the blood fraction of choice. Fasting

plasma glucose values in excess of 120 mg% (true glucose)

are considered indicative of diabetes mellitus; values

between 110-120 mg% are equivocal and should be

confirmed with a GTT. The 2 hours postprandial (PP) test

should be done instead of fasting glucose levels. Emotional

hyperglycemia from secretion of epinephrine as well as

cerebral lesions (skull fractures, tumors, vascular accident,

and encephalitis) and carbon monoxide poisoning, often

provoke hyperglycemia and glycosuria, must be considered

in the evaluation of blood glucose measurements.

Two-hours Postprandial Blood Glucose

After an overnight fast (12 hours), the patient is given

a breakfast of 100 g of carbohydrate or a 100 g glucose

load. Previous to the test, the patient should have been

on an adequate carbohydrate diet (300 g daily) and all

medications that influence glucose tolerance should

have been discontinued 3 days prior to the test. Two

hours later [2 hours PP or PC, post cibum] a single blood

sample is withdrawn for analysis. A value within normal

limits makes the diagnosis of diabetes mellitus unlikely,

plasma glucose values in the range of 120-140 mg% are

suspicious and in excess of 140 mg% (true glucose),

diagnosis is most likely and should be confirmed by GTT.

The limitations of a single 2 hours PP glucose value

include the following:

1. Slow absorption, which may delay the peak.

2. Rapid absorption with early hyperglycemia, rapid

fall in concentration of blood glucose (due to insulin

release), and then a second hyperglycemic peak

due to the effects of counter regulatory responses

(epinephrine, glucagon, growth hormone).

3. Errors in timing specimen collection.

Diagnosis and Classification of Diabetes

Mellitus: New Criteria

New recommendations for the classification and diagnosis

of diabetes mellitus include the preferred use of the terms

“type 1” and “type 2” instead of “IDDM” and “NIDDM”

to designate the two major types of diabetes mellitus:

simplification of the diagnostic criteria for diabetes

mellitus to two abnormal fasting plasma determinations:

and a lower cutoff for fasting plasma glucose [126 mg per

dL (7 mmol per L) or higher] to confirm the diagnosis of

diabetes mellitus. These changes provide an easier and

more reliable means of diagnosing persons at risk of

complications from hyperglycemia. Currently, only one

half of the people who have diabetes mellitus have been

diagnosed. Screening for diabetes mellitus should begin at

45 years of age and should be repeated every three years in

persons without risk factors, and should begin earlier and

be repeated every 3 years in persons without risk factors,

and should begin earlier and be repeated more often in

those with risk factors. Risk factors include obesity, firstdegree relatives with diabetes mellitus, hypertension,

hypertriglyceridemia or previous evidence of impaired

glucose homeostasis. Earlier detection of diabetes mellitus

may lead to tighter control of blood glucose levels and a

reduction in the severity of complications associated with

this disease.

Diabetes mellitus is a group of metabolic disorders

with one common manifestation: hyperglycemia. Chronic

hyperglycemia causes damage to the eyes, kidneys, nerves,

heart and blood vessels. The etiology and pathophysiology

436 Concise Book of Medical Laboratory Technology: Methods and Interpretations leading to the hyperglycemia, however, are markedly

different among patients with diabetes mellitus, dictating

different prevention strategies, diagnostic screening

methods and treatments. The adverse impact of

hyperglycemia and the rationale for aggressive treatment

have recently been reviewed.

In June 1997, an international expert committee released

a report with new recommendations for the classification

and diagnosis of diabetes mellitus. These new recommendations were the result of more than two years of collaboration

among experts from the American Diabetes Association

and the World Health Organization (WHO). The use of

classification systems and standardized diagnostic criteria

facilitates a common language among patients, physicians,

other healthcare professionals and scientists.

Previous Classification

In 1979, the National (American) Diabetes Data Group

produced a consensus document standardizing the

nomenclature and definitions for diabetes mellitus.

This document was endorsed one year later by WHO.

The two major types of diabetes mellitus were given

names descriptive of their clinical presentation: “insulindependent diabetes mellitus” (IDDM) and “noninsulindependent diabetes mellitus” (NIDDM).

Diabetes mellitus that is characterized by absolute insulin deficiency

and acute onset, usually before 25 years of age, should now be

referred to as type 1 (not type I, IDDM or juvenile) diabetes mellitus.

However, as treatment recommendations evolved, correct classification of the type of diabetes mellitus became

confusing. For example, it was difficult to correctly classify

persons with NIDDM who were being treated with insulin.

This confusion led to the incorrect classification of a large

number of patients with diabetes mellitus complicating

epidemiologic evaluation and clinical management. The

discovery of other types of diabetes with specific pathophysiology that did not fit into this classification system

further complicated the situation. These difficulties, along

with new insights into the mechanisms of diabetes mellitus, provided a major impetus for the development of a

new classification system.

The National Diabetes Data Group also established

the oral glucose tolerance test (using a glucose load

of 75 g) as the preferred diagnostic test for diabetes

mellitus. However, this test has poor reproducibility, lacks

physiologic relevance and is a weaker indicator of longterm complications compared with other measures of

hyperglycemia. Furthermore, many high-risk patients are

unwilling to undergo this time-consuming test on a repeatbasis. The new diagnostic criteria also address this issue.

Changes in the Classification System

The new classification system identifies four types of

diabetes mellitus: type 1, type 2, other specific types and

gestational diabetes. Arabic numerals are specifically used

in the new system to minimize the occasional confusion of

type “II” as the number “11”. Each of the types of diabetes

mellitus identified extends across a clinical continuum of

hyperglycemia and insulin requirements.

Any patient with two fasting plasma glucose levels of 126 mg per dL

(7.0 mmol per L) or greater is considered to have diabetes mellitus.

Type 1 diabetes mellitus (formerly called type I, IDDM or

juvenile diabetes) is characterized by beta cell destruction

caused by an autoimmune process, usually leading to

absolute insulin deficiency. The onset is usually acute,

developing over a period of a few days to weeks. Over 95%

of persons with type 1 diabetes mellitus develop the disease

before the age of 25, with an equal incidence in both sexes

and an increased prevalence in the white population.

A family history of type 1 diabetes mellitus, gluten

enteropathy (celiac disease) or other endocrine disease

is often found. Most of these patients have the “immunemediated form” of type 1 diabetes mellitus with islet cell

antibodies and often have other autoimmune disorders,

such as Hashimoto’s thyroiditis, Addison’s disease, vitiligo

or pernicious anemia. A few patients, usually those of

African or Asian origin, have no antibodies but have

a similar clinical presentation; consequently, they are

included in this classification and their disease is called

the “idiopathic form” of type 1 diabetes mellitus.

Type 2 diabetes mellitus (formerly called NIDDM,

type  II or adult-onset) is characterized by insulin

resistance in peripheral tissue and an insulin secretory

defect of the beta cell. This is the most common form of

diabetes mellitus and is highly associated with a family

history of diabetes, older age, obesity and lack of exercise.

It is more common in women, especially women with

a history of gestational diabetes. Insulin resistance and

hyperinsulinemia eventually lead to impaired glucose

tolerance. Defective beta cells become exhausted, further

fueling the cycle of glucose intolerance and hyperglycemia.

The etiology of type 2 diabetes mellitus is multifactorial

and probably genetically based, but it also has strong

behavioral components.

The etiologic classifications of diabetes mellitus are

listed in Table 17.1.

Diabetes Mellitus: Laboratory Diagnosis 437

TABLE 17.1: Etiologic classification of diabetes mellitus

Types of diabetes mellitus of various known etiologies

are grouped together to form the classification called

“other specific types.” This group includes persons with

genetic defects of beta-cell function (this type of diabetes

was formerly called MODY or maturity-onset diabetes

in youth) or with defects of insulin action; persons with

diseases of the exocrine pancreas, such as pancreatitis or

cystic fibrosis; persons with dysfunction associated with

other endocrinopathies (e.g. acromegaly); and persons

with pancreatic dysfunction caused by drugs, chemicals or

infections.