Sodium tolbutamide (1 g is given IV in 20 mL of saline

over 2 minutes to a (12 hours) fasting subject. A fasting

preinjection blood specimen and postinjection samples

at 2, 5, 10, 20, 30, 60, 120, and 180 minutes are collected

for glucose estimation. Juvenile diabetics (insulinopenic)

reveal virtually no response, while adult (maturity-onset)

diabetics show a delayed increase in blood glucose concentrations. Patients with an insulin-secreting tumor (islet cell

adenoma or hyperplasia) reveal a profound depression

of blood glucose values, which persists below 50 mg% at

2 hours, this is associated with maximum insulin values

as early as 15 minutes. Appropriate medical precautionary measures must be readily available (sterile glucose

injection) and used promptly with any stress tolerance test

whenever a patient’s condition warrants intervention and

cessation of test.

IV Insulin Tolerance Test

Here insulin 0.1 unit/kg of ideal body weight is

administered IV in a fasting state, blood specimens are

collected at appropriate intervals over a 2 hour period

for glucose analysis. Within 30 minutes the blood glucose

concentration falls to about 50 or 60% of the fasting level

and returns to normal fasting levels between 1 hour

30 minutes, and 2 hours. A failure to observe such a

depression in blood glucose concentration may imply

insulin resistance. This may be occasionally seen in adult

type diabetes, as well as in acromegaly and Cushing’s

syndrome. In panhypopituitarism and adrenocortical

insufficiency (Addison’s disease) a more profound and

sustained decrease in blood glucose may be observed,

hence caution should be exercised in patients suspected

of having these disorders.

Glycosylated Hemoglobin (HbA1c); Glycohemoglobin

(GHb); Diabetic Control Index

Kits Available Commercially

Increased

Diabetes mellitus, glycosuria, and hyperglycemia.

Decreased

See, below, Factors that affect results.

Description

Glycosylated hemoglobin is blood glucose bound to

hemoglobin (Hb) and includes from HbA1a, HbA1b, and

HbA1c. HbA1c is formed as hemoglobin, is gradually

glycosylated throughout the 120 days; red blood cell

lifespan, and forms the largest portion of the three

Diabetes Mellitus: Laboratory Diagnosis 443

glycosylated Hb fractions. The amount of glycosylated

hemoglobin found and stored in erythrocytes depends on

the amount of glucose available. HbA1c is a reflection of

how well blood glucose levels have been controlled for up

to the prior 4 months. Hyperglycemia in diabetics if usually

a cause of an increase in HbA1c.

Factors that Affect Results

a. Reject hemolyzed specimens.

b. Falsely increased values may be due to fetal-maternal

transfusion, hemodialysis, hereditary persistence of

fetal hemoglobin, neonates and pregnancy.

c. Falsely decreased values may be due to anemia

(hemolytic, pernicious, sickle cell); chronic loss of

blood ; effects of splenectomy; renal failure (chronic);

and thalassemias.

Other Data

a. Glycosylated hemoglobin cannot be used to monitor

control of diabetic clients with chronic renal failure,

as levels are significantly lower due to shortened

erythrocyte survival.

Approximately 8.5% of total hemoglobin: HbA1

Glycohemoglobin is one of the types of minor

hemoglobins found in every individual. Hemoglobin A,

undergoes change or glycosylation to hemoglobin A1a, A1b,

A1c by a slow, nonenzyme process within the RBCs during

their circulating lifespan of 120 days. Simply putting it,

glycohemoglobin is blood glucose bound to hemoglobin.

The RBC, as it circulates, combines, some of the glucose

from the bloodstream with its own content of hemoglobin

to form glycohemoglobin in a one-way reaction. The

amount of glycosylated hemoglobin found and stored

by the RBC depends on the amount of glucose available

to it over the RBCs 120 days lifespan. In diabetics with

hyperglycemia, the increase in GHb is usually caused

by an increase in HbA1c. The glucose concentration will

increase when hyperglycemia caused by insulin deficiency

develops. This glycosylation is irreversible.

Test Significance

This test is an index of long-term glucose control. GHb

monitoring reflects the average blood sugar level for the

2 to 3 months period before the test. The more glucose

the RBC is exposed to, the higher the percentage of GHb.

The test provides vital information about the success of

treatment of diabetes such as the adequacy of dietary

or insulin therapy, allows determination of duration of

hyperglycemia in new cases of juvenile onset diabetes with

acute ketoacidosis, provides a sensitive estimate of glucose

imbalance in mild cases of diabetes, and is an evaluation of

effectiveness of old and new forms of therapy such as oral

hypoglycemic agents, single or multiple insulin injections,

and B-cell transplantation. Test results are not affected

by time of day, meal intake, exercise, just administered

diabetic drugs, emotional stress, patient cooperation or

accuracy.

The estimation of GHb is of greater importance

for specific groups of patients. These groups include

diabetic children, diabetics in whom the renal threshold

for glucose is abnormal, unstable insulin-dependent

diabetics in whom blood sugars vary markedly from day

to day, patients who do not test urine regularly for glucose,

and people who, before their scheduled appointments,

will change their usual habits, dietary or otherwise, so that

their metabolic control appears better than it actually is.

Clinical Relevance

1. Values are increased in poorly controlled and newly

diagnosed diabetes. In these instances, HbA1c levels

comprise 8 to 12% of the total hemoglobin.

2. With optimal insulin control, the HbA1c levels return

toward normal.

3. A diabetic patient who has only recently come under

good control may still have a high concentration of

glycosylated hemoglobin. This level will only gradually

decline as newly formed RBCs with nearly normal GHb

replace older RBCs with high concentrations of GHb.

Interfering Factors

1. Spurious results should be expected in every case of

hemoglobinopathy distinguishable from hemoglobin

A by electrophoresis.

2. Decreased value in pregnancy and sickle cell anemia,

increased value in thalassemia.

Confusion in interpretation of results may occur

because there are two tests for determining glycosylated

hemoglobin. The most specific test measures HbA1, which

includes hemoglobin A1a, A1b and A1c. There are different

expected values for each test. Keep in mind that HbA1, is

always 2% to 4% higher than HbA1c.

GLYCOSYLATED HEMOGLOBIN KIT (ION

EXCHANGE RESIN METHOD) FOR THE

QUANTITATIVE DETERMINATION OF

GLYCOHEMOGLOBIN IN BLOOD (FOR IN VITRO

DIAGNOSTIC USE ONLY)

(Courtesy: Tulip Group of Companies)

Summary

Glycosylated hemoglobin (GHb) is formed continuously

by the adduction of glucose by covalent bonding to the

444 Concise Book of Medical Laboratory Technology: Methods and Interpretations aminoterminal valine of the hemoglobin beta chain

progressively and irreversibly over a period of time and

is stable till the life of the RBC. This process is slow, nonenzymatic and is dependent on the average blood glucose

concentration over a period of time.

A single glucose determination reflects the glucose

level at the time. GHb on the other hand reflects the mean

glucose level over an extended period of time. Thus GHb

reflects the metabolic control of glucose level over a period

of time unaffected by diet, insulin, other drugs, or exercise

on the day of testing. GHb is now widely recognized as an

important test for the diagnosis of diabetes mellitus and is

a reliable indicator of the efficacy of therapy.

Principle

Glycosylated hemoglobin (GHb) has been defined

operationally as the fast fraction hemoglobins HbA1

(Hb A1a, A1b, A1c) which elute first during column

chromatography. The nonglycosylated hemoglobin, which

consists of the bulk of hemoglobin, has been designated

HbA0.