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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.