Clinical Chemistry 481

Causes of Monoclonal Gammopathies

¾ Multiple myeloma

¾ Waldenstrom’s macroglobulinemia

¾ Benign idiopathic monoclonal gammopathy

¾ Heavy chain diseases

¾ Collagen disorders, autoimmune diseases

¾ Certain lymphomas

¾ Cirrhosis liver

¾ Neoplasms of colon, prostate, breast, female genital

tract, stomach and lungs

¾ Myeloproliferative disorders-CML, polycythemia,

myelofibrosis, erythrimic myelosis, erythroleukemia,

other acute leukemias

¾ Aberrations in lipid metabolism

¾ Diabetes mellitus.

Interfering Factors

1. Low levels of albumin occur normally in all trimester’s

of pregnancy.

2. Bromosulfalein may cause a false elevation. Therefore,

a serum protein test should not be done within 48 hours

following a BSP test.

3. See appendix for complete listing of drugs that

interfere with total protein levels.

SERUM CHOLESTEROL

Cholesterol (CHOD/PAP Method)

(Courtesy: Tulip Group of Companies)

For the determination of cholesterol in serum or plasma

(for in vitro diagnostic use only).

Summary

Cholesterol is the main lipid found in blood, bile and brain

tissues. It is the main lipid associated with arteriosclerotic

vascular diseases. It is required for the formation of

steroids and cellular membranes. The liver metabolizes

the cholesterol and it is transported in the blood

stream by lipoproteins. Increased levels are found in

hypercholesterolemia, hyperlipidemia, hypothyroidism,

uncontrolled diabetes, nephrotic syndrome, and cirrhosis.

Decreased levels are found in malabsorption, malnutrition, hyperthyroidism, anemias and liver diseases.

Principle

Cholesterol esterase hydrolyzes esterified cholesterols to

free cholesterol. The free cholesterol is oxidised to form

hydrogen peroxide which further reacts with phenol and

4-aminoantipyrine by the catalytic action of peroxidase to

form a red colored quinoneimine dye complex. Intensity

of the color formed is directly proportional to the amount

of cholesterol present in the sample.

 Cholesterol esterase

Cholesterol esters + H2O Cholesterol + Fatty acids

 Cholesterol oxidase

Cholesterol + O2 Cholestenone + H2O2

 Peroxidase

H2O2 + 4 Aminoantipyrine + Phenol Red

quinoneimine

dye + H2O

Normal Reference Values

Serum/plasma (Suspicious) : 220 mg/dL and above

(Elevated) : 260 mg/dL and above

It is recommended that each laboratory establish its

own normal range representing its patient population.

Contents 2 × 75 mL 2 × 150 mL

L1: Enzyme reagent 1 2 × 60 mL 2 × 120 mL

L2: Enzyme reagent 2 2 ×15 mL 2 × 30 mL

S: Cholesterol standard (200 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: Pour the contents of 1 bottle of L2

(Enzyme reagent 2) into 1 bottle of L1 (Enzyme reagent

1). This working reagent is stable for at least 8 weeks when

stored at 2–8°C. Upon storage the working reagent may

develop a slight pink color however, this does not affect the

performance of the reagent. Alternatively for flexibility as

much of working reagent may be made as and when desired

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

1 part of L2 (Enzyme reagent 2). 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.

Sample Material

Serum, EDTA plasma. Cholesterol is reported to be stable

in the sample for 7 days when stored at 2–8°C. The sample

should preferably be of 12 to 14 hours fasting.

482 Concise Book of Medical Laboratory Technology: Methods and Interpretations Procedure

Wavelength/filter : 505 nm (Hg 546 nm)/green

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)

Working reagent 1.0 1.0 1.0

Distilled water 0.01 - -

Cholesterol standard (S) - 0.01

Sample - - 0.01

Mix well and incubate at 37°C for 5 minutes or at RT (25°C)

for 15 minutes. Measure the absorbance of the standard

(Abs S), and test sample (Abs T) against the blank, within

60 minutes.

Calculations

 Abs T

Cholesterol in mg/dL = ________ × 200 Abs S

Linearity

This procedure is linear upto 750 mg/dL. If the value

exceeds this limit, dilute the serum with normal saline

(NaCL 0.9%) and repeat the assay. Calculate the value

using the proper dilution factor.

Note

Anticoagulants such as fluorides and oxalates result in

false low values. The test is not influenced by Hb values

upto 20 mg/dL and bilirubin upto 10 mg/dL.

System Parameters

Reaction : End point Interval : ...

Wavelength : 505 nm Sample

volume

: 0.01 mL

Zero setting :  Reagent blank Reagent

volume

: 1.00 mL

Incubation

temperature

: 37°C / RT Standard : 200 mg/dL

Incubated

time

: 5 min/15 min Factor :

Delay time : — React slope : Increasing

Read time : — Linearity : 750 mg/dL

No. of read : — Units : mg/dL

Normal values

Male Female

SI units SI units

Age mg/L mmol/L mg/dL mmol/L

Total cholesterol

Adult

20–24 124–218 3.21–5.64 122–216 3.16–5.59

25–29 133–244 3.44–6.32 128–222 3.32–5.75

30–34 138–254 3.57–6.58 130–230 3.37–5.96

35–39 146–270 3.78–6.99 140–242 3.63–6.27

40–44 151–268 3.91–6.94 147–252 3.81–6.53

45–49 158–276 4.09–7.15 152–265 3.94–6.86

50–54 158–277 4.09–7.17 162–285 4.20–7.38

55–59 156–276 4.04–7.15 172–300 4.45–7.77

60–64 159–276 4.12–7.15 172–297 4.45–7.69

65–69 158–274 4.09–7.10 171–303 4.43–7.85

> 70 144–265 3.73–6.86 173–280 4.48–7.25

Child

Cord blood 44–103 1.14–2.66 50–108 1.29–2.79

< 4 114–203 2.95–5.25 112–200 2.90–5.18

5–9 121–203 3.13–5.25 126–205 3.26–5.30

10–14 119–202 3.08–5.23 124–201 3.21–5.20

15–19 113–197 2.93–5.10 119–200 3.08–5.18

High-density lipoprotein cholesterol (HDL)

Adult

20–24 30–63 0.78–1.63 33–79 0.85–2.04

25–29 31–63 0.80–1.63 37–83 0.96–2.15

30–34 28–63 0.72–1.63 36–77 0.93–1.99

35–39 29–62 0.75–1.60 34–82 0.88–2.12

40–44 27–67 0.70–1.73 34–88 0.88–2.28

45–49 30–64 0.78–1.66 34–87 0.88–2.25

50–54 28–63 0.72–1.63 37–92 0.96–2.38

55–59 28–71 0.72–1.84 37–91 0.96–2.35

60–64 30–74 0.78–1.91 38–92 0.98–2.38

65–69 30–75 0.78–1.94 35–96 0.91–2.48

> 70 31–75 0.80–1.94 33–92 0.85–2.38

Child

Cord blood 6–53 0.16–1.37 13–56 0.34–1.45

5–9 38–75 0.98–1.94 36–73 0.93–1.89

10–14 37–74 0.96–1.91 37–70 0.96–1.81

15–19 30–63 0.78–1.63 35–74 0.91–1.91

Contd...

Clinical Chemistry 483

Low-Density lipoprotein Cholesterol (LDL)

Adult

20–24 66–147 1.71–3.81 57–159 1.48–4.12

25–29 70–165 1.81–4.27 71–164 1.84–4.25

30–34 78–185 2.02–4.79 70–156 1.81–4.04

35–39 81–189 2.10–4.90 75–172 1.94–4.45

40–44 87–186 2.25–4.92 74–174 1.92–4.51

45–49 97–202 2.51–5.23 79–186 2.05–4.82

50–54 89–197 2.31–5.10 88–201 2.28–5.21

55–59 88–203 2.28–5.26 89–210 2.31–5.44

60–64 83–210 2.15–5.44 100–224 2.59–5.80

65–69 98–210 2.54–5.44 92–221 2.38–5.72

> 70 88–186 2.28–4.82 96–206 2.49–5.34

Child

Cord

blood 20–56 0.52–1.45 21–58 0.54–1.50

5–9 63–129 1.63–3.34 68–140 1.76–3.63

10–14 64–133 1.66–3.44 68–136 1.76–3.52

15–19 62–130 1.61–3.37 59–137 1.53–3.55

SI Units

Cholesterol

esters

60–75% of total or 0.60–0.75

< 210 mg/dL < 5.43 mmol/L

Free

cholesterol

< 50 mg/dL < 1.29 mmol/L

LDL:HDL

ratio

< 3 < 3

Clinical Relevance

1. Increased levels of cholesterol

 a. Levels above 250 mg/dL are considered elevated

and call for a triglyceride test.

 b. Conditions related to elevated cholesterol

 1. Cardiovascular disease and atherosclerosis

 2. Type II, familial hypercholesterolemia

 3. Obstructive jaundice (also an increase in

bilirubin)

 4. Hypothyroidism (decreased in hyperthyroidism)

 5. Nephrosis

 6. Xanthomatosis

 7. Uncontrolled diabetes

 8. Nephrotic syndrome

 9. Obesity.

 c. Free versus esterified cholesterol.

 There is a markedly abnormal ratio of free to esterified

cholesterol in disease of the liver biliary tract,

infectious disease, and extreme cholesterolemia.

2. Decreased levels of cholesterol

 a. Conditions where cholesterol is not absorbed

from the gastrointestinal tract

 1. Malabsorption

 2. Liver disease

 3. Hyperthyroidism

 4. Anemia

 5. Sepsis

 6. Stress

 7. Drug therapy such as antibiotics.

 b. Other disorders related to decreased cholesterol

levels

 1. Pernicious anemia

 2. Hemolytic jaundice

 3. Hyperthyroidism

 4. Severe infections

 5. Terminal stages of debilitating diseases such as

cancer

 6. Hypolipoproteinemias.

 c. Esterol fraction decreases in liver diseases,

liver cell injury, malabsorption syndrome, and

malnutrition.

3. Increased levels of cholesterol esters are associated

with familial deficiency of Lecithin—cholesterol

acyltransferase (LCAT).

4. Decreased levels of cholesterol are associated with

liver disease. This is because persons with liver

diseases may have impaired formation of LCAT with

a resulting deficiency of the enzyme.

5. Cholesterol ester storage disease causes accumulation

of cholesterol esters in the tissues, but it has no effect

on the percentage of esterified cholesterol in the

blood.

6. The higher the cholesterol phospholipid ratio, the

greater the possible risk of developing atherosclerosis.

Interfering Factors

1. Cholesterol is normally slightly elevated in pregnancy.

2. Estrogen decreases plasma cholesterol and oophorectomy increases it.

3. Many drugs may cause a change in the blood cholesterol

Patient Preparation

1. Advise patient about fasting for a night for 12 hours

before the test.

2. Water is permitted.

3. Before fasting, the patient should be on a normal diet

for 7 days before testing.

Contd...

484 Concise Book of Medical Laboratory Technology: Methods and Interpretations 4. No alcohol should be consumed 24 hours before

testing.

5. Lipid lowering drugs such as estrogen, oral contraceptives,

and salicylates should be withheld.

 Calculate the change in absorbance ∆A for both the

standard and test.

For standard ∆AS = A2S – A1S

For test ∆AT = A2T – A1T

Calculations

 ∆AT

Creatinine in mg/dL = ________ × 2.0 ∆AS

 ∆AT

Urine creatinine in g/L = _______ × 1.0

 ∆AS

Urine creatinine =

 Urine creatinine in g/L × _______________________________

g/24 hours Volume of urine in liters 24 hours

Linearity

The procedure is linear upto 20 mg/dL of Creatinine. If

values exceed this limit, dilute the sample with distilled

water and repeat the assay. Calculate the value using the

proper dilution factor.

Note

The buffer reagent may turn milky or show white

precipitates at cold temperatures. This is not a deterioration of the reagent. Dissolve/clear the same by warming

the reagent to 37°C with gentle swirling before use. The

determination is not specific and may be affected by the

presence of large quantities of reducing substances.

Clinical Chemistry 475

As the test is temperature sensitive, it is essential to

maintain the indicated reaction timings and temperatures

meticulously during the test procedure.

System Parameters

Reaction : Fixed time kin Interval : 60 seconds

Wavelength : 520 nm Sample

volume

: 0.10 mL

Zero setting :  Distilled water Reagent

volume

: 1.00 mL

Incubation

temperature

: 30°C/37°C Standard : 2 mg/dL

Incubated

time

: - Factor : -

Delay time : 30 seconds React slope : Increasing

Read time : 60 seconds Linearity : 20 mg/dL

No. of read : 2 Units : mg/dL

Clinical Relevance

Causes of Raised Serum Creatinine Levels

All renal causes of uremia are usually associated with raised

serum creatinine values. Elevated BUN levels in a patient

with normal creatinine usually signal a nonrenal cause

for the uremia. With severe, permanent renal damage,

urea levels continue to climb, but creatinine values tend

to plateau. At very high creatinine levels, some is excreted

across the alimentary tract.

Decreased Creatinine Levels Occur in

Muscular dystrophy.

Interfering Factors

1. High levels of ascorbic acid can give a falsely increased

level.

2. Drugs influencing kidney function (diuretics and

dextran), chloral hydrate, marijuana, acetohexamide,

guanethidine, furosemide, chloramphenicol,

and sulfonamides can cause a change in blood

creatinine.

3. A diet high is roast meat will cause increased levels.

4. Many drugs may cause a change in the blood creatinine.

 A normal blood serum creatinine does not always

indicate unimpaired renal function. A normal value

cannot be used as standard for a patient who is known

to have existing renal disease.

Serum Bilirubin

Normal Values

SI units

Total bilurubin

1 month – adult < 1.5 mg/dL 1.7–20.5 µmol/L

Premature infant

Cord < 2.8 mg/dL < 48 µmol/L

24 hours 1–6 mg/dL 17–103 µmol/L

48 hours 6–8 mg/dL 103–137 µmol/L

3–5 days 10–12 mg/dL 171–205 µmol/L

Full-term infant

Cord < 2.8 mg/dL < 48 µmol/L

24 hours 2–6 mg/dL 34–103 µmol/L

48 hours 6–7 mg/dL 103–120 µmol/L

3–5 days 4–6 mg/dL 68–103 µmol/L

Direct bilirubin 0.0–0.3 mg/dL 1.7–5.1 µmol/L

Indirect bilirubin 0.1–1.0 mg/dL 1.7–17.1 µmol/L

Bilirubin (Mod Jendrassik and Grof’s Method)

(Courtesy: Tulip Group of Companies)

For the determination of direct and total bilirubin in serum

(for in vitro diagnostic use only).

Summary

Bilirubin is mainly formed from the heme portion of aged

or damaged RBCs. It then combines with albumin to form

a complex which is not water soluble. This is referred to

as indirect or unconjugated bilirubin. In the liver, this

bilirubin complex is combined with glucuronic acid into a

water soluble conjugate. This is referred to as conjugated

or direct bilirubin. Elevated levels of bilirubin are found in

liver diseases (hepatitis, cirrhosis), excessive hemolysis/

destruction of RBC (hemolytic jaundice) obstruction

of the biliary tract (obstructive jaundice) and in drug

induced reactions. The differentiation between the direct

and indirect bilirubin is important in diagnosing the cause

of hyperbilirubinemia.

Principle

Bilirubin reacts with diazotized sulfanilic acid to form

a colored azobilirubin compound. The unconjugated

bilirubin couples with the sulfanilic acid in the presence of

a caffein-benzoate accelerator. The intensity of the color

476 Concise Book of Medical Laboratory Technology: Methods and Interpretations formed is directly proportional to the amount of bilirubin

present in the sample.

Bilirubin + Diazotized Sulfanilic acid→ Azobilirubin

Compound

Normal Reference Values

Serum (Direct) : upto 0.2 mg/dL

(Total) : upto 1.0 mg/dL

It is recommended that each laboratory establish its

own normal range representing its patient population.

Contents 30 tests 75 tests

L1: Direct bilirubin reagent 75 mL 150 mL

L2: Direct nitrite reagent 4 mL 4 mL

L1: Total bilirubin reagent 75 mL 150 mL

L2: Total nitrite reagent 4 mL 4 mL

S : Artificial standard (10 mg/dL) 10 mL 10 mL

Storage/Stability

All reagents are stable at RT till the expiry mentioned on

the label.

Reagent Preparation

Reagents are ready to use. Do not pipette with mouth.

Sample Material

Serum. Bilirubin is reported to be stable in the sample for

4 days at 2–8°C protected from light as it is photosensitive.

Procedure

Wavelength/filter : 546 nm/yellow-green

Temperature : RT

Light path : 1 cm

Direct Bilirubin Assay

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

Test (T):

Addition

Sequence

B

(mL)

T

(mL)

Direct bilirubin reagent (L1) 1.0 1.0

Direct nitrite reagent (L2) - 0.05

Sample 0.1 0.1

Mix well and incubate at RT for exactly 5 minutes.

Measure the absorbance of the test samples (Abs T)

immediately against their respective blanks.

Total Bilirubin Assay

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

test (T):

Addition

Sequence

B

(mL)

T

(mL)

Total bilirubin reagent (L1) 1.0 1.0

Total nitrite reagent (L2) - 0.05

Sample 0.1 0.1

Mix well and incubate at RT for 10 min. Measure the

absorbance of the test samples (Abs T) immediately

against their respective blanks.

Calculations

Total or direct bilirubin in mg/dL = Abs T × 13 (13 being

the factor).

Linearity

This procedure is linear upto 20 mg/dL. If values exceed

this limit, dilute the sample with distilled water and repeat

the assay. Calculate the value using the proper dilution

factor.

Note

In case, the exact wavelength is not available the artificial

standard (S) may be used. Measure the absorbance of

the artificial standard against distilled water with the

appropriate filter and keep the same for future calculations

by dividing the Abs T with the Abs. of the Std. × 10. Discard

the artificial standard after use.

In case of neonates where the sample quantity is a

limitation, and the samples have high bilirubin (above

3 mg/dL), only 0.05 mL/0.02 mL of the sample may be

used for bilirubin estimation. The calculation factor in

this case would be 24.9/60.5 respectively instead of 13. In

case of using the standard the value of the same would be

19.1/46.5 mg/dL respectively instead of 10 mg/dL.

System Parameters

Reaction : End point Interval :

Wavelength : 546 nm Sample

volume

: 0.10 mL

Zero setting : Sample blank Reagent

volume

: 1.05 mL

Incubation

temperature

: RT Standard :

Incubated time : 5 min/10 min Factor : 13

Delay time : — React slope : Increasing

Read time : — Linearity : 20 mg/dL

No. of read : — Units : mg/dL

Clinical Chemistry 477

Causes of Hyperbilirubinemia

Unconjugated (Indirect) Hyperbilirubinemia

I. Overproduction of bilirubin

 A. Hemolytic disorders.

 1. Congenital (e.g. hemoglobinopathies)

 2. Acquired (e.g. Coombs’ positive anemia)

 3. Liver disease (e.g. hepatitis and cirrhosis).

 B. Shunt hyperbilirubinemia

II. Defective uptake and storage of bilirubin

 A. Idiopathic unconjugated hyperbilirubinaemia.

 1. Hereditary-Gilbert’s syndrome.

 2. Acquired

 – Post-viral hepatitis.

 – Post-portacaval shunt.

 B. Decreased availability of cytoplasmic binding

proteins (Y and Z) in newborn and premature infants.

 C. Drugs (e.g. flavispidic acid).

III. Defective glucuronyl transferase activity.

 A. Deficiency.

 1. In newborn and premature infants

 2. Crigler-Najjar syndrome.

 B. Inhibition

 1. Abnormal steroids in breast milk or maternal

plasma (Lucey-Driscoll type).

 2. Drugs (e.g. novobiocin).

Conjugated (Direct) Hyperbilirubinemia

Defective excretion of conjugated bilirubin

A. Hereditary

 1. Dubin-Johnson syndrome

 2. Rotor syndrome.

B. Obstructive

 1. Intrahepatic cholestasis

 a. Cirrhosis (occasionally)

 b. Hepatitis (often)

 c. Alcoholic liver disease (occasionally)

 d. Drugs (e.g. chlorpromazine and methyltestosterone).

 e. Primary biliary cirrhosis.

 2. Extrahepatic obstruction.

 a. Gallstones

 b. Carcinoma of the bile duct, pancreas, ampulla

of Vater

 c. Bile duct stricture

 d. Biliary atresia.

Interfering Factors

1. A 1 hour exposure of the specimen to sunlight or

high intensity artificial light at room temperature will

reduce the bilirubin content.

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.