HDL CHOLESTEROL

PEG/CHOD-PAP Method

(Courtesy: Tulip Group of Companies)

For the determination of HDL cholesterol in serum or

plasma (for in vitro diagnostic use only).

Summary

Lipoproteins are the proteins which mainly transport fats in

the bloodstream. They can be grouped into chylomicrons,

very low density lipoproteins (VLDL), low density

lipoproteins (LDL) and high density lipoproteins (HDL).

Chylomicrons and VLDL transport mainly triglycerides,

though VLDLs also transport some amount of cholesterol.

LDL carries cholesterol to the peripheral tissues where it

can be deposited and increase the risk of arteriosclerotic

heart and peripherial vascular disease. Hence, high levels

of LDL are atherogenic. HDL transports cholesterol from

the peripherial tissues to the liver for excretion, hence,

HDL has a protective effect. The measurement of total

and HDL cholesterol and triglycerides provide valuable

information for the risk assessment of coronary heart

diseases.

Principle

When the serum is reacted with the polyethylene glycol

contained in the precipitating reagent, all the VLDL and

LDL are precipitated. The HDL remains in the supernatant

and is then assayed as a sample for cholesterol using the

cholesterol (CHOD/PAP) reagent.

Normal Reference Values

Prognostically

favorable

Standard

risk level

Risk

indicator

HDL cholesterol

(mg/dL)

Males

Females

> 55

> 65

35–55 < 35

45–65 < 45

LDL cholesterol

(mg/dL)

Males

Females

< 150 150–190 > 190

Total cholesterol

HDL cholesterol

Males

Females

> 3.8

> 3.1

3.8–5.9

3.1–4.6

< 5.9

< 4.6

It is recommended that each laboratory establish its

own normal range representing its patient population.

Contents 75 mL

L1 : Enzyme reagent 1 60 mL

L2 : Enzyme reagent 2 15 mL

L3 : Pricipitating reagent 2.5 mL

S : HDL cholesterol standard (25 mg/dL) 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 and HDL cholesterol

are reported to be stable in serum for 7 days when stored

at 2–8°C. The sample should preferably be of 12 to 14 hours

fasting.

Procedure

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

Temperature : 37°C/RT

Light path : 1 cm

Precipitation of VLDL and LDL

Pipette into a clean dry test tube :

Precipitating reagent (L3) 0.1 mL

Sample 0.1 mL

Mix well and incubate at RT for 5 minutes. Centrifuge at

2500-3000 rpm to obtain a clear supernatant.

Cholesterol Assay

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

standard (S), and test (T):

Clinical Chemistry 485

Addition

Sequence

B

(mL)

S

(mL)

T

(mL)

Working reagent 1.0 1.0 1.0

Distilled water 0.05 - -

HDL standard (S) - 0.05 -

Supernatant * - - 0.05

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.

* If only total cholesterol is to be determined use only

0.01 mL of DW/cholesterol Std/sample directly in the

cholesterol assay.

Calculations

 Abs. T

HDL cholesterol in mg/dL = _______ × 25 × 2 Abs. S

(Where 2 is the dilution factor due to the deproteinization step)

Calculation of LDL Cholesterol (mg/dL)

(Friedewald’s Formula)

 Triglycerides

= Total cholesterol _ (_____________ (

_ HDL cholesterol 5

Friedewald’s formula is reliable provided that:

1. No chylomicrons are present, i.e. it is a fasting sample.

2. Triglyceride values are below 400 mg/dL.

3. Type III hyperlipoproteinemia is absent.

Linearity

This procedure is linear upto 150 mg/dL of HDL cholesterol.

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

The supernatant should be clear. If it is hazy or cloudy,

the sample should be diluted 1 + 1 with normal saline

(NaCL 0.9%) and the precipitation step should be repeated

(Results × 2)

Anticoagulants such as fluoride, oxalates and hemolyzed

serums should not be used.

System Parameters

Reaction : End Point Interval : -

Wavelength : 505 nm Sample

volume

: 0.05 mL

Zero setting : Reagent blank Reagent

volume

: 1.00 mL

Incubation

temperature

: 37°C/RT Standard : 25 mg/dL × 2

Incubated

time

: 5 min/15 min Factor : -

Delay time : - Reaction

slope

: Increasing

Read time : - Linearity : 150 mg/dL

No. of read : - Units : mg/dL

Risk Factor

Coronary heart disease (CHD) risk factor can be calculated

using total lipid profile, as suggested by Castelli, et al. The

risk factor gives a most accurate and definite assessment of

heart disease risk.

The factors are calculated by the ratio of total cholesterol

to HDL—cholesterol and by the ratio of LDL—cholesterol

(Low density lipoproteins—cholesterol) to HDL—

cholesterol.

Risk Ratio: Total/ Ratio: LDL/

HDL__Cholesterol HDL__Cholesterol

Men Women Men Women

½ Average 3.43 3.27 1.00 1.47

Average 4.97 4.44 3.55 3.22

2 × Average 9.55 7.05 6.25 5.03

3 × Average 23.99 11.04 7.99 6.14

HDL Cholesterol ppt Set (PEG Precipitation Method)

(Courtesy: Tulip Group of Companies)

For the determination of HDL cholesterol in serum or

plasma (for in vitro diagnostic use only).

Summary

Lipoproteins are the proteins which mainly transport fats in

the bloodstream. They can be grouped into chylomicrons,

very low density lipoproteins VLDL, low density lipoproteins

(LDL) and high density lipoproteins (HDL). Chylomicrons

and VLDL transport mainly triglycerides, though VLDLs

also transport some amount of cholesterol. LDL carries

cholesterol to the peripheral tissues where it can be deposited

and increase the risk of arteriosclerotic heart and peripheral

vascular disease. Hence, high levels of LDL are atherogenic.

HDL transports cholesterol from the peripheral tissues to

the liver for excretion, hence HDL has a protective effect. The

Contd...

Contd...

486 Concise Book of Medical Laboratory Technology: Methods and Interpretations measurement of total and HDL cholesterol and triglycerides

provide valuable information for the risk assessment of

coronary heart diseases.

Principle

When the serum is reacted with the polyethylene glycol

contained in the precipitating reagent, all the VLDL and

LDL are precipitated. The HDL remains in the supernatant

and is then assayed as a sample for cholesterol using the

cholesterol (CHOD/PAP) reagent.

Normal Reference Values

Prognostically standard Risk

favourable risk level indicator

HDL cholesterol

(mg/dL)

Males

Females

> 55

> 65

35–55

45–65

< 35

< 45

LDL cholesterol Males < 150

Females

150–190 > 190

Total cholesterol

HDL cholesterol

Males > 3.8 3.8–5.9 < 5.9

Females > 3.1 3.1–4.6 < 4.6

It is recommended that each laboratory establish its

own normal range representing its patient population.

Contents 10 mL

L1: Precipitating reagent 10 mL

S: HDL cholesterol standard (25 mg/dL) 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.

After the precipitation step cholesterol reagent is

required additionally for conducting the cholesterol assay.

Sample Material

Serum, EDTA plasma. HDL cholesterol is reported to

be stable in serum for 7 days when stored at 2–8°C. The

sample should preferably be of 12 to 14 hours fasting.

Procedure

Precipitation of VLDL and LDL:

Pipette into a clean dry test tube

Precipitating reagent (L1) 0.1 mL

Sample 0.1 mL

Mix well and incubate at RT for 5 minutes. Centrifuge at

2500–3000 rpm to obtain a clear supernatant.

Procedure for the Cholesterol Assay

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 B S T

Sequence (mL) (mL) (mL)

Working reagent 1.0 1.0 1.0

Distilled water 0.05 - -

HDL standard (S) - 0.05 -

Supernatant - - 0.05

Mix well and incubate at 37°C for 5 mm. 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

HDL cholesterol in mg/dL = _______ × 25 × 2 Abs S

(Where 2 is the dilution factor due to the deproteinization

step)

Calculation of LDL cholesterol (mg/dL):

(Friedewald’s formula)

 Triglycerides = (Total cholesterol) _ _______________

5 _ (HDL cholesterol)

Freidewald’s formula is reliable provided that:

1. No chylomicrons are present, i.e. it is a fasting sample.

2. Triglyceride values are below 400 mg/dL.

3. Type III hyperlipoproteinemia is absent.

Linearity

This procedure is linear upto 150 mg/dL of HDL cholesterol. 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

The supernatant should be clear. If it is hazy or cloudy,

the sample should be diluted 1 + 1 with normal saline

(NaCL 0.9%) and the precipitation step should be repeated

(results × 2).

Anticoagulants such as fluoride, oxalates and hemolyzed

serums should not be used.

Clinical Chemistry 487

System Parameters

Reaction : End point Interval :

Wavelength : 505 nm Sample

volume

: 0.05 mL

Zero setting :  Reagent

blank

Reagent

volume

: 1.00 mL

Incubation

temperature

: 37°C / RT Standard : 25 mg/dL × 2

Incubated

time

: 5 min/15 min Factor

Delay time : — React slope : Increasing

Read time : — Linearity : 150 mg/dL

No. of read : — Units : mg/dL

Clinical Relevance

1. Increased values are associated with a chronic liver

disorder.

 

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.

 

2. Contrast media 24 hours before measurement may

cause an altered reaction.

3. A high fat meal may cause decreased bilirubin levels

by interfering with the clinical reactions.

4. Air bubbles and shaking of the specimen may cause

decreased levels.

5. Foods (carrots, etc.) and drugs increase the yellowish

hue in the serum.

6. Refer to the Many drugs can interfere with Bilirubin

tests for a listing of the many drugs that may interfere

with testing for bilirubin.

7. Hemolyzed blood will falsely elevate bilirubin level.

Comments

1. In severe obstructive jaundice with formation of

biliverdin, low results for the degree of jaundice will

be obtained since biliverdin does not react with the

diazo reagent and cannot be determined.

2. An unusual source of otherwise unexplained elevated

serum bilirubin has been described following 48 hours

fasting. A normal bilirubin value from 0.68 mg% may

rise to the abnormal range at 1.87 mg%.

Icterus Index

The icterus index is a measure of the degree of icterus

(yellowish-green color) in a plasma or serum specimen

in cases of jaundice. This is just a screening test for

hyperbilirubinemia. Substances other than bilirubin

in the serum (carotene, xanthophyll, hemoglobin, etc.)

may contribute to the icterus index, therefore, limiting its

clinical utility. The test is now considered to be obsolete.

Reagents

A. Potassium dichromate solution

 1. Stock solution (1%)

 Dissolve 1 g of potassium dichromate in 70 mL of

water placed in a 100 mL volumetric flask. Add 2

drops of sulfuric acid and dilute to 100 mL mark

with distilled water. Store in a glass-stoppered

brown/amber colored bottle.

 2. Working standard solution (0.1%)

 Pipette 10 mL of the stock solution into a 100 mL

volumetric flask and dilute to 100 mL mark with

distilled water.

B. Saline (0.9% NaCL) isotonic.

Method

A. Dilute the serum specimen ten times with saline (1 mL of

serum mixed with 9 mL of saline) in a test tube and mix.

B. Transfer the diluted serum into a cuvette and read

absorbance at 420 to 460 nm. If too dark, dilute further

478 Concise Book of Medical Laboratory Technology: Methods and Interpretations and multiply the final reading with the dilution factor

utilized here.

C. Determine the icterus index from the calibration curve.

Multiply the result by dilution factor. If the serum is

diluted ten times, the dilution factor is 10.

Calibration Curve

A. Prepare three concentrations of the standard by diluting appropriate quantities of the stock solution of

potassium dichromate in three 100 mL volumetric

flasks

 1. Five mL stock mixed with 95 mL of water (1:20).

This corresponds to 5 units.

 2. 25 mL of stock solution made to 100 mL with water

(1:4 dilution). This corresponds to 25 units.

 3. 50 mL of stock solution made to 100 mL with water

(1:2 dilution). This corresponds to 50 units.

B. Read the absorbance of each working standard

solution corresponding to 5, 25 and 50 units at 420 to

460 nm using water as blank.

C. Tabulate the results with the units of icterus index and

the corresponding absorbance values.

D. Plot a calibration curve and use this for the determination of icterus index.

TOTAL PROTEINS

Biuret Method

(Courtesy: Tulip Group of Companies)

For the determination of total proteins in serum and

plasma (for in vitro diagnostic use only).

Summary

Proteins are constituents of muscle, enzymes, hormones

and several other key functional and structural entities

in the body. They are involved in the maintenance of the

normal distribution of water between blood and the tissues.

Consisting mainly of albumin and globulin the fractions

vary independently and widely in diseases. Increased

levels are found mainly in dehydration. Decreased levels

are found mainly in malnutrition, impaired synthesis,

protein losses as in hemorrhage or excessive protein

catabolism.

Principle

Proteins, in an alkaline medium, bind with the cupric ions

present in the biuret reagent to form a blue-violet colored

complex. The intensity of the color formed is directly

proportional to the amount of proteins present in the

sample.

Proteins + Cu++→ Blue violet colored complex

Normal Reference Values

Serum and plasma : 6.0–8.0 g/dL

It is recommended that each laboratory establish its

own normal range representing its patient population.

Contents 150 mL 2 × 150 mL

Carton 1

L1: Biuret reagent 150 mL 2 × 150 mL

Carton 2

S: Protein standard ( 8 g/dL) 5 mL 5 mL

Storage/Stability

Carton 1 : Biuret reagent is stable at RT till the expiry

mentioned on the label.

Carton 2 : Protein standard is stable at 2–8°C till the

expiry mentioned on the label.

Reagent Preparation

Reagents are ready to use. Protect from bright light.

Sample Material

Serum or plasma. Proteins are reported to be stable in the

sample for 6 days at 2–8°C.

Procedure

Wavelength/filter : 550 nm (Hg 546 nm)/yellow-green

Temperature : RT/37°C

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)

Biuret reagent (L1) 1.0 1.0 1.0

Distilled water 0.02

Protein standard (S) - 0.02

Sample - - 0.02

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

for 30 minutes. Measure the absorbance of the standard

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

60 minutes.

Calculations

 Abs T

Total proteins in g/dL = _________ = × 8 Abs S

Linearity

This procedure is linear upto 15 g/dL. If values exceed

this limit, dilute the sample with distilled water and

Clinical Chemistry 479

repeat the assay. Calculate the value using the proper

dilution factor.

Note

Do not use if the reagent shows turbidity or black

precipitates.

System Parameters

Reaction : End point Interval :

Wavelength : 550 nm Sample vol : 0.02 mL

Zero setting : Reagent blank Reagent vol : 1.00 mL

Incubation

temperature

: 37°C/RT Standard : 8 g/dL

Incubated

time

: 10 mm/30 min Factor :

Delay time : React slope : Increasing

Read time : Linearity : 15 g/dL

No. of read : Units : g/dL

SERUM ALBUMIN

Determination of Serum Albumin (BCG Method)

(Courtesy: Tulip Group of Companies)

For the determination of albumin in serum or plasma (for

in vitro diagnostic use only).

Summary

Albumin consists of approximately 60% of the total

proteins in the body, the other major part being globulin.

It is synthesized in the liver and maintains the osmotic

pressure in blood. Albumin also helps in the transportation

of drugs, hormones and enzymes. Elevated levels are

rarely seen and are usually associated with dehydration.

Decreased levels are seen in liver diseases (hepatitis,

cirrhosis). Malnutrition, kidney disorders, increased fluid

loss during extensive burns and decreased absorption in

gastrointestinal diseases.

Principle

Albumin binds with the dye bromocresol green in a

buffered medium to form a green colored complex. The

intensity of the color formed is directly proportional to the

amount of albumin present in the sample.

Albumin + Bromocresol green→ Green albumin BCG

complex.

Normal Reference Values (Albumin)

Serum, plasma (albumin) : 3.7–5.3 g/dL

Globulin : 2.3–3.6 g/dL

A/G Ratio : 1.0–2.3

It is recommended that each laboratory establish its

own normal range representing its patient population.

Contents 150 mL 2 × 150 mL

Carton 1

L1: BCG reagent 150 mL 2 × 150 mL

Carton 2

S: Albumin standard (4 g/dL) 5 mL 5 mL

Storage/Stability

Carton 1 : BCG reagent is stable at RT till the expiry

mentioned on the label.

Carton 2 : Albumin Standard is stable at 2–8°C till the

expiry mentioned on the label.

Reagent Preparation

Reagents are ready to use. Protect from bright light.

Sample Material

Serum, EDTA plasma. Albumin is reported to be stable in

the sample for 6 days at 2–8°C.

Procedure

Wavelength/filter : 630 nm (Hg 623 nm)/Red

Temperature : 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)

BCG reagent (L1) 1.0 1.0 1.0

Distilled water 0.01 - -

Albumin Standard (S) - 0.01 -

Sample - - 0.01

Mix well and incubate at RT for 5 minutes. Measure

absorbance of the standard (Abs S), and test sample (Abs T)

against the blank.

Calculations

 Abs T

Albumin in g/dL = ________ × 4 Abs S

Globulin in g/dL = (Total proteins) — (Albumin)

 (in g/dL) (in g/dL)

 Albumin in g/dL A/G Ratio = _________________

 Globulin in g/dL

Linearity

The procedure is linear upto 7 g/dL. If values exceed this

limit, dilute the sample with distilled water and repeat

480 Concise Book of Medical Laboratory Technology: Methods and Interpretations the assay. Calculate the value using the proper dilution

factor.

Note

Gross hemolysis, ampicillin and heparin interfere with

the results. Elevated bilirubin and lipemic samples may

have a slight effect on accuracy. For grossly lipemic

samples run a sample blank by adding 0.02 mL sample

in 2 mL distilled water. Read the absorbance against

DW and substract the blank absorbance from the test

absorbance.

System Parameters

Reaction : End point Interval :

Wavelength : 630 nm Sample

volume

: 0.01 mL

Zero setting :  Reagent

blank

Reagent

volume

: 1.00 mL

Incubation

temperature

: RT Standard : 4 g/dL

Incubated time : 5 minutes Factor :

Delay time : — React slope : Increasing

Read time : — Linearity : 7 g/dL

No. of read : —- Units : g/dL

Normal Values

Total Proteins

SI units

Adults 6.0–8.0 g/dL 60–80 g/L

Children

Premature 4.3–7.6 g/dL 43–76 g/L

Newborn 4.6–7.4 g/dL 46–74 g/L

Infant 6.0–6.7 g/dL 60–67 g/L

Child 6.2–8.0 g/dL 62–80 g/L

Specimen Collection and storage

1. Serum is the specimen of choice.

2. Avoid excessive hemolysis since every 100 mg/dL

of hemoglobin corresponds to about 100 mg/dL of

albumin.

3. Albumin in serum is reported stable for one week at

room temperature (18–30°C) and approximately one

month when stored in the refrigerator (2–8°C) and

protected against evaporation.

Clinical Relevance

Causes of Hypoalbuminemia

Reduced synthesis

¾ Malnutrition

¾ Malabsorption syndromes

¾ Chronic inflammatory diseases

¾ Acute hepatitis (lasting 14 days or more)

¾ Chronic liver disease

¾ Genetic abnormalities.

Increased Loss

¾ Nephrotic syndrome

¾ Massive burns

¾ Protein-losing enteropathy.

Increased catabolism

¾ Massive burns

¾ Widespread malignancy.

Multifactorial

¾ Cirrhosis

¾ Congestive heart failure

¾ Pregnancy.

Increased albumin levels are generally not observed

(When albumin concentration decreases there is a relative

increase in globulins. However, there is a definite rise in

globulins in mono/polyclonal gammopathies).

Disorders Associated with Polyclonal

Gammopathies

Chronic liver disease

¾ Nutritional cirrhosis

¾ Primary biliary cirrhosis

¾ Chronic active hepatitis

¾ Viral hepatitis.

Collagen diseases

¾ Rheumatoid arthritis

¾ Systemic lupus erythematosus

¾ Sjögren’s syndrome

¾ Felty’s syndrome

¾ Polymyositis

¾ Scleroderma.

Chronic Infections

¾ Tuberculosis

¾ Osteomyelitis

¾ Deep fungi

¾ Syphilis

¾ Bronchitis.

Miscellaneous

¾ Metastatic carcinoma

¾ Cystic fibrosis

¾ Recovery from trauma.