A hemolyzed preparation of whole blood is mixed

continuously for 5 minutes with a weakly binding cationexchange resin. The labile fraction is eliminated during

the hemolysate preparation and during the binding.

During this mixing, HbA0 binds to the ion exchange

resin leaving GHb free in the supernatant. After the

mixing period, a filter separator is used to remove the

resin from the supernatant. The percent glycosylated

hemoglobin is determined by measuring absorbances of

the glycosylated hemoglobin (GHb) fraction and the total

hemoglobin (THb) fraction. The ratio of the absorbances

of the Glycosylated hemoglobin and the Total hemoglobin

fraction of the Control and the Sample is used to calculate

the percent Glycosylated hemoglobin of the sample.

Normal Reference Values

Normal : 4.5–8.0%

Good control : 8.0–9.0%

Fair control : 9.0–10.0%

Poor control : > 10.0%.

It is recommended that each laboratory establish its

own normal range representing its patient population.

Contents 10 Tests 25 Tests

Ion Exchange Resin 10 × 3 mL 25 × 3 mL

(Predispensed Tubes)

Lysing Reagent 5 mL 12.5 mL

Control (10% GHb) 1 × 1 mL 1 × 1 mL

Resin Separators 10 Nos. 25 Nos.

Storage/Stability

Contents stable at 2–8oC till the expiry mentioned on the

label. Do not freeze. The Resin separators can be removed

on opening the kit and stored at RT.

Reagent Preparation

The ion exchange resin tubes and the lysing reagent are

ready to use.

Reconstitute the control with 1 mL of distilled water.

Allow to stand for 10 min with occasional mixing. The

reconstituted control is stable for at least 7 days when

stored at 2-8oC tightly sealed, and at least 4 weeks when

stored at -20oC. Do not thaw and refreeze.

Sample Material

Whole blood. Preferably fresh and collected in EDTA. GHb

in whole blood is reported to be stable for one week at

2-8oC.

Procedure

Wavelength/Filter : 415 nm (Hg 405 nm)

Temperature : RT

Light path : 1 cm

A. Hemolysate preparation

1. Dispense 0.5 mL lysing reagent into tubes labeled as control (C) and test (T).

2. Add 0.1 mL of the reconstituted control and

well-mixed blood sample into the appropriately

labeled tubes. Mix until complete lysis is evident.

3. Allow to stand for 5 minutes.

B. Glycosylated hemoglobin (GHb) Separation

1. Remove cap from the ion-exchange resin tubes

and label as control and test.

2. Add 0.1 mL of the hemolysate from Step A into

the appropriately labeled Ion exchange resin

tubes.

3. Insert a resin separator into each tube so that the

rubber sleeve is approximately 1 cm above the

liquid level of the resin suspension.

4. Mix the tubes on a rocker, rotator or a vortex

mixer continuously for 5 minutes.

5. Allow the resin to settle, then push the resin

separator into the tubes until the resin is firmly

packed.

6. Pour or aspirate each supernatant directly into

a cuvette and measure each absorbance against

distilled water.

C. Total hemoglobin (THb) fraction

1. Dispense 5.0 mL of distilled water into tubes

labeled as control and test.

Diabetes Mellitus: Laboratory Diagnosis 445

2. Add to it 0.02 mL of hemolysate from Step A into

the appropriately labeled tube. Mix well.

3. Read each absorbance against distilled water.

Calculations

Ratio of Control (RC) = Abs. Control GHb

Abs. Control THb

Ratio of Test (RT) = Abs. Test GHb

Abs. Test THb

GHb in % = Ratio of Test (RT)

Ratio of Control (RC)

× 10 (Value of Control)

Linearity

The glycosylated hemoglobin procedure shows linearity

for GHb levels in the range of 4.0–20.0%.

Notes

Blood samples with hemoglobin greater than 18 g/dL

should be diluted 1 + 1 with normal saline before the assay.

Samples from patients with hemoglobinopathies,

decreased red cell survival times, gross lipemia may show

incorrect results.

Do not use ion exchange resin tubes in case of turbidity

or visible discoloration.

Diabetics with metabolic imbalance may have

extremely high levels of the labile aldimine form. In such

cases the incubation time during hemolysate preparation

may be increased to 15 minutes to ensure elimination of

this instable fraction.

For mean blood glucose level based upon GHbA1/

HbA1c refer to Table 17.4.

Insulin

Normal Values

SI Units

Adult

Fasting level < 17 µU/mL 42–243 pmol/L

or 1.00 mg/L

Newborn 3–20 µU/mL 21–139 pmol/L

Infant < 13 µU/mL < 89 pmol/L

Prepubertal child < 13 µU/mL < 89 pmol/L

Panic levels > 30 µU/mL > 290 pmol/L

Last trimester, amniotic fluid 11.3 µU/mL 78 pmol/L

Insulin is a hormone produced in pancreas by the beta

cells of the islets of Langerhans, regulates the metabolism

of carbohydrates along with liver, adipose, muscle, and

other target cells and is responsible for maintaining a

constant level of blood glucose. The rate of insulin secretion

is determined primarily by the level of blood glucose

perfusing the pancreas and is affected by hormonal status,

the autonomic nervous system, and nutritional status.

Test Significance

This measurement of the insulin secretory response to

glucose may be of value in establishing the diagnosis of

insulinoma and in the evaluation of abnormal carbohydrate

and lipid metabolism. Insulin levels are also helpful in

supporting the diagnosis of diabetes in persons with

borderline abnormalities of the GTT. This determination

is invaluable in the investigation of fasting hypoglycemic

patients and may be useful in the differentiation of islet cell

neoplasms. Insulin levels may be ordered along with GTT.

Clinical Relevance

Increased values are associated with:

A. Insulinoma: diagnosis of insulinoma is based on

1. Association of insulinoma with hypoglycemia

2. Persistent hypoglycemia along with hyperinsulinemia between 2 and 3 hours after injection of

tolbutamide.

3. Failure of C-peptide suppression when plasma

glucose is 40 mg/dL or less. After 100 g of glucose,

normal insulin will rise less than 2 µU/ml to 25 to

231 in half hour, 18 to 276 in one hour, 16 to 166

in 2 hours, 4 to 38 in 3 hours. The results may be

too variable to be of diagnostic importance.

B. Acromegaly

C. Cushing’s syndrome.

Interfering Factors

Falsely increased values are associated with food intake,

obesity, and use of oral contraceptives.

(Method: see Endocrinology chapter).

C-Peptide

Normal Values

SI Units

Qualitative Negative

Quantitative

Adult 68–8200 ng/mL 68–8200 µg/L

or 20 mg/dL

or < 8 µg/mL

Cord blood 10–350 ng/mL 10–350 µg/L

446 Concise Book of Medical Laboratory Technology: Methods and Interpretations TABLE 17.4: For the conversion of glycosylated hemoglobin A1 (GHbA1) to gylcosylated hemoglobin A1c (HbA1c) and to the mean blood

glucose level (MBG)

GHbA1 HbA1c MBG GHbA1 HbA1c MBG GHbA1 HbA1c MBG GHbA1 HbA1c MBG

5.0 3.46 —- 9.0 6.81 141 13.0 10.16 252 17.0 13.51 —-

5.1 3.54 —- 9.1 6.89 144 13.1 10.25 255 17.1 13.60 —-

5.2 3.63 —- 9.2 6.98 146 13.2 10.33 258 17.2 13.68 —-

5.3 3.71 —- 9.3 7.06 149 13.3 10.41 261 17.3 13.68 —-

5.4 3.79 —- 9.4 7.15 152 13.4 10.50 264 17.4 13.85 —-

5.5 3.88 —- 9.5 7.23 155 13.5 10.58 266 17.5 13.93 —-

5.6 3.96 —- 9.6 7.31 158 13.6 10.66 269 17.6 14.02 —-

5.7 4.04 —- 9.7 7.40 160 13.7 10.75 272 17.7 14.10 —-

5.8 4.13 —- 9.8 7.48 163 13.8 10.83 275 17.8 14.18 —-

5.9 4.21 —- 9.9 7.56 166 13.9 10.92 278 17.9 14.27 —-

6.0 4.30 57 10.0 7.65 169 14.0 11.00 280 18.0 14.35 —-

6.1 4.38 60 10.1 7.73 171 14.1 11.08 —- 18.1 14.44 —-

6.2 4.46 63 10.2 7.82 174 4.2 11.17 —- 18.2 14.52 —-

6.3 4.55 65 10.3 7.90 177 14.3 11.25 —- 18.3 14.60 —-

6.4 4.63 68 10.4 7.98 180 14.4 11.34 —- 18.4 14.69 —-

6.5 4.71 71 10.5 8.07 183 14.5 11.42 —- 18.5 14.77 —-

6.6 4.80 74 10.6 8.15 185 14.6 11.50 —- 18.6 14.85 —-

6.7 4.88 77 10.7 8.23 188 14.7 11.59 —- 18.7 14.94 —-

6.8 4.97 79 10.8 8.32 191 14.8 11.67 —- 18.8 15.02 —-

6.9 5.05 82 10.9 8.40 194 14.9 11.75 —- 18.9 15.11 —-

7.0 5.13 85 11.0 8.49 197 15.0 11.84 —- 19.0 15.19 —-

7.1 5.22 88 11.1 8.57 199 15.1 11.92 —- 19.1 15.27 —-

7.2 5.30 91 11.2 8.65 202 15.2 12.01 —- 19.2 15.36 —-

7.3 5.39 93 11.3 8.74 205 15.3 12.09 —- 19.3 15.44 —-

7.4 5.47 96 11.4 8.82 208 15.4 12.17 —- 19.4 15.53 —-

7.5 5.55 99 11.5 8.91 211 15.5 12.26 —- 19.5 15.61 —-

7.6 5.64 102 11.6 8.99 213 15.6 12.34 —- 19.6 15.69 —-

7.7 5.72 104 11.7 9.07 216 15.7 12.42 —- 19.7 15.78 —-

7.8 5.80 107 11.8 9.16 219 15.8 12.51 —- 19.8 15.86 —-

7.9 5.89 110 11.9 9.24 222 15.9 12.59 —- 19.9 15.94 —-

8.0 5.97 113 12.0 9.32 224 16.0 12.68 —- 20.0 16.03 —

8.1 6.06 116 12.1 9.41 227 16.1 12.76 —-

8.2 6.14 118 12.2 9.49 230 16.2 12.84 —-

8.3 6.22 121 12.3 9.58 233 16.3 12.93 —-

8.4 6.31 124 12.4 9.66 236 16.4 13.01 —-

8.5 6.39 127 12.5 9.74 238 16.5 13.09 —-

8.6 6.47 130 12.6 9.83 241 16.6 13.18 —-

8.7 6.56 132 12.7 9.91 244 16.7 13.26 —-

8.8 6.64 135 12.8 9.99 247 16.8 13.35 —-

8.9 6.73 138 12.9 10.08 250 16.9 13.43 —-

MBG in mg/dL = 33.3 x HbA1c value—86

These values are linear in the range of 6.5–13% of HbA1c values

Diabetes Mellitus: Laboratory Diagnosis 447

C-peptide is formed during the conversion of proinsulin

to insulin in the beta cells of the pancreas. It is secreted

into the bloodstream in almost equal concentration with

insulin. Normally, a strong correlation exists between

levels of insulin and C-peptide, except possibly in obese

subjects and in the presence of islet cell tumors.

Test Significance

C-peptide level measurement provides a reliable indication

of beta and secretory function and insulin secretions.

This determination has its most useful application in the

evaluation of endogenous secretion of insulin when the

presence of circulatory insulin antibodies interferes with

the direct assay of insulin. This situation is most likely to

occur in diabetics who have been treated with bovine pork

insulin. The test is also useful in evaluating hypoglycemic

states in identifying surreptitious injection of insulin,

and in confirmation of remission of diabetes mellitus.

Furthermore, monitoring following pancreatectomy for

removal of cancer can provide a means of detecting the

presence of residual tissue.

Clinical Relevance

1. Increased values are associated with endogenous

hyperinsulinism in insulin-dependent diabetic

persons when a high level of insulin is also present.

2. Decreased levels are associated with persons who have

been surreptitiously injecting insulin and who have

both hypoglycemia and high insulin levels.

3. Normal levels are found in persons who have had a

remision of diabetes mellitus.

 (Method: see Endocrinology chapter).

Glucagon

Normal Values

1. 50-200 pg/mL plasma

2. Glucagon response in normal people after a standard

test meal of carbohydrates, fat and protein is a gradual

increase from 92 plus or minus 12 pg/mL to a peak of

125 plus or minus 13 pg/mL.

3. In a glucose tolerance test, glucagon levels will

significantly decline from fasting levels during the

hyperglycemic first hour in normal people.

Glucagon is a peptide hormone produced by alpha cells

of the islets of Langerhans in the pancreas. In the liver,

this hormone promotes glucose production. This action

of glucagon is opposed to that of insulin. The normal

coordinated release patterns of this hormone provide a

sensitive control mechanism for glucose production and

storage. For example, low glucose levels result in release,

whereas conditions of hyperglycemia reduce circulating

glucagon levels to approximately 50% of the amount in the

fasting state.

Kidneys play an important role in the metabolism of

glucagon.

Abnormally high levels of glucagon recede once insulin

therapy begins to control diabetes, and levels slowly revert

to normal in persons on maintenance doses of insulin.

Also, in contrast to the normal glucagon, secretion in

diabetics does not decrease following ingestion of a

carbohydrate meal. However, an arginine infusion causes

greatly increased glucagon secretion in normal persons.

Test Significance

This measurement has clinical significance in two ways.

Glucagon deficiency reflects a general loss of pancreatic

tissue. Compelling evidence for glucagon deficiency

is the failure of glucagon levels to rise during arginine

infusion. Hyperglucagonemia occurs in diabetics, acute

pancreatitis, and in situations where catecholamine

secretion is greatly augmented as in pheochromocytoma

and in the presence of infection.

Clinical Relevance

1. Increased levels are associated with:

a. Acute pancreatitis.

b. Diabetes mellitus. Persons with severe diabetic

ketoacidosis are reported to have levels five times

normal fasting levels despite marked hyperglycemia.

c. Glucagonoma.

d. Uremia.

e. Infections.

f. Pheochromocytoma.

2. Reduced levels are associated with:

a. Inflammatory disease with loss of pancreatic

tissue.

b. Neoplastic replacement of pancreas.

c. Surgical removal of pancreas.

Interfering Factors

Increased levels occur in vigorous exercise and in trauma.

Other Important Tests in Diabetics

Urine — Ketone bodies (present in diabetic

ketoacidosis).

Serum — Cholesterol Can be assessed

 (raised) chemically or

 — Triglycerides } by serum

 (raised) electrophoresis

 — Ketones (raised in presence of ketonuria).

448 Concise Book of Medical Laboratory Technology: Methods and Interpretations Hypoglycemia

By definition means blood glucose levels less than 50 mg%.

Causes of Hypoglycemia

Spontaneous (fasting) Hypoglycemia

1. Excessive insulin

• Insulinoma or insulin-secreting carcinoma

• Erythroblastosis fetalis.

2. Non-endocrine tumor__retroperitoneal fibroma

3. Glycogen storage disease of the liver

4. Malnutrition or malabsorption

5. Adrenocortical or pituitary failure

6. Liver necrosis

7. Hereditary galactosemia

8. Reye’s syndrome and other forms of ketotic

hypoglycemia in children.

Induced Hypoglycemia

1. Excessive insulin:

• Overtreated insulin

• Leucine (includes some islet cell tumors)

• Sulfonyl ureas

• Functional:

– Prediabetic

– Postgastrectomy.

– Hemodialysis with hypertonic glucose

– Idiopathic.

2. Reduced gluconeogenesis.

• Ethanol

• Hypoglycin

• Hereditary fructose intolerance

• Failure of glucagon secretion.

3. Persistent increase of peripheral glucose uptake:

• Failure of catecholamine secretion

• Propranolol blockade of catecholamine effect.

4. Cause uncertain

• Pentamidine.

RAPID DIAGNOSTICS

1. Urine sugar: See urinalysis chapter for dipstick tests.

2. Blood sugar: Various instant blood glucose meters are

available.

Accu-Chek®

(Courtesy: Roche Diagnostics)

Accu-Chek Active System: Virtually Painfree Testing

in 5 Seconds

Things to do. Places to go. Whatever pace you live your life

at, new Accu-Chek Active is with you all the way. In just 5

seconds, Accu-Chek Active delivers highly accurate results,

whenever and wherever you need them. It’s the quickest,

best-looking system ever. If you don’t want diabetes to

slow you down, it’s definitely the way to go.

Accu-Chek Softclix (Fig. 17.1)

The exclusive Accu-Chek Softclix Lancing Device with its

11 variable depth settings and lancet allows you to draw

the minimum amount of blood required.

¾ Virtually pain-free testing

¾ Small, discreet pen-like design

¾ Eleven variable depth settings for maximum comfort

¾ Lancets available on prescription.

Accu-Chek Active Meter

¾ Small, sleek design

¾ Inserting test strip switches on meter automatically

¾ Two hundred test memory with date and time for

automatic recording of results.

Active Glucose Test Strips

¾ Only a tiny drop of blood required

¾ Accurate results in just 5 seconds.

Running a Quality Control Test

For the quality control test, please have the following items

ready (Fig. 17.2):

¾ Your Accu-Chek Active meter with the coding chip

inserted

¾ The pack of Accu-Chek Active Glucose test strips you

took the coding chip from

¾ The Accu-Chek Active Control solutions

¾ Carefully read the pack inserts that came with the test

strips and the control solutions, and select a control

solution

FIG. 17.1: Accu-Chek Softclix

Diabetes Mellitus: Laboratory Diagnosis 449