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.