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.

 

Operator Errors

Errors of the operator can be minimized by the practice

of good technique in the laboratory preparation of the

solutions and in the operation of the instrument. The

operator should be guided in the latter instance by the

instructions provided by the manufacturer. An awareness,

of the sources of error in the preceding categories should

tend to reduce these errors.

CLINICAL CHEMISTRY

Specimen Collection and Processing

With the exception of glucose, triglycerides and inorganic

phosphorus, most blood chemical constituents reveal no

significant change after a standard breakfast, so it is not

essential for the patient to be in an absolute fasting state

prior to blood specimen collection. However, lipemia

(lactescence), caused by transient rise in triglycerides

as chylomicrons following a meal containing fat may

cause interference with a large number of chemical

determinations because of turbidity. Therefore, blood

is always collected from a patient in the post-absorptive

state. This can be accomplished with an overnight fast

(12–14 hours, especially for lipids), although a 4 to 6 hours

fast will usually suffice.

Venipuncture should be performed for obtaining blood.

Disposable needles eliminate the hazard of serum hepatitis

transmission. Heparin is the most ideal anticoagulant for

plasma determination. The cost is quite prohibitive so

others EDTA, and trisodium citrate can be used without

significant alteration in reading and results. For glucose,

oxalate-fluoride mixture is used. Fluoride impairs

glycolysis of the blood cells. Prompt separation of plasma/

serum is essential to yield a proper specimen for most

chemical estimations. Always collect a little more blood

than required so as not to fall short of it subsequently. For

1 mL of serum about 2.5 mL of blood should be withdrawn.

Labelling and identification is important.

Pipettes: For dispensing test materials or reagents, etc. exact

quantities are needed. For volumes till 0.1 mL Borosil’s

pipettes can be used. Otherwise autopipettes for micro to

macro sampling can be used. Dispensing exact amounts

of test samples/reagents is the first step to accurate clinical

chemistry.

Proper Specimen Collection

If the commercially available kits are being used, follow

the manufacturers guidelines and collect the requisite

amount of blood.

Specimen Collection

Chemistry (plain tube)

Amylase Lipase

Alcohol Lithium

Bilirubin LATS and TSH

Barbiturate Triglyceride

Salicylate Electrolytes

BSP BUN

Calcium Uric acid

Cholesterol

Copper

Creatinine

CPK

SGOT

SGPT

Urea

T4, T3, TSH

Iron and iron binding

capacity

LDH

Chemistry (Heparin)

pH

Ammonia

RBC Potassium

Renin

Plasma testosterone

Cholinesterase

Plasma cortisol

Methemoglobin

Plasma hemoglobin

Chemistry

Oxalate, fluoride tube

Glucose

Glucose tolerance test

466 Concise Book of Medical Laboratory Technology: Methods and Interpretations Hematology (EDTA)

Complete blood counts

WBC, RBC, Hb, PCV,

MCV, MCH, MCHC

Differential count

Absolute eosinophil count

Hematology (EDTA)

Hb electrophoresis

G6PD screening

Reticulocyte count

ESR

Sickling test

Platelet count.

Hematology (plain tube)

Haptoglobin,

LE preparation

Serum viscosity.

Hematology (Sodium citrate)

PTTK

Prothrombin time

Thrombin time

Fibrinogen titer

Fibrinogen level.

Blood bank (plain tube)

Crossmatch

Typing

Coombs’ test

Antibody identification.

Serology (plain tube)

α1 antitrypsin

Antinuclear antibody

Antistreptolysin-O

Antithyroid antibodies

Ceruloplasmin

C-reactive protein

Cold agglutinins

Paul Bunnel test

Immunoglobulins

Leptospira agglutination test

VDRL

Australia antigen.

Processing

Ideally all measurements should be performed within

1 hour after collection. Tests where proteins are first

precipitated with tungstic acid, trichloroacetic acid or

barium sulfate—samples for these tests can be stored in

a refrigerator at 4–6°C if the interval before the analysis

exceeds 30 minutes. In medical chemistry, plasma can be

used for virtually all measurements (ideal anticoagulant

being heparin), although a few require serum (serum

enzymes and protein electrophoresis), while whole blood

can for all practical purposes be eliminated. Whenever a

delay of more than 1 hour is anticipated, refrigerate the

sample at 4oC. For extracting serum—let the blood clot at

room temperature (takes about 20–30 minutes), loosen the

clot at the top by a stick. Centrifuge blood for 10 minutes

at 3,000 rpm, serum can be removed with the use of a

pasteur pipette. Label and store the serum in a refrigerator

at 4–6°C until analyzed or freeze at –20°C, if the analysis is

to be delayed by more than 4 hours.

Centrifuge

While centrifuging the principle of balance must always be

observed. Tubes of equal weight, shape, and size should be

placed in opposing positions in the centrifuge head (using

water filled tubes whenever necessary). Tubes should be

supported by appropriately shaped rubber cushions in the

carrier of the centrifuge head. The speed of the centrifuge

should be slowly accelerated.

Difficulties

1. All tubes should be chemically clean, i.e. free of actual

or potential organic and/or inorganic constituents that

may alter the result of a chemical analysis. They need

not be sterile.

2. Hemolysis: It should always be avoided as release of RBC

contents (e.g. LDH, acid phosphatase and potassium)

or through color change (especially for photometric

measurements using shorter wavelengths of the visible

spectrum 400–500 nm) results may be falsely high.

Hemoglobin interferes with specific chemical reactions

(e.g. diazotization inhibition in bilirubin estimation).

Blood Collection, Precautions and Errors

1. Excessive venous stasis by prolonged application

of tourniquet should be avoided. This would also

raise concentration of certain constituents of blood

hormones, calcium, K+, Lactic acid, etc.).

2. The syringe, needle and the tube should be moisture

free.

3. Blood should be withdrawn by needle of gauge less

than 21.

4. Expelling blood through the needle into the container

should be avoided.

Clinical Chemistry 467

5. Do not shake blood in container to mix with anticoagulants. Mix by gentle repetitive inversion—about

6 to 8 times.

 

 but in most instruments the intensity is below the

danger level. Methods in which photochemical reactions

are likely to occur usually mention the precautions to be

taken against light exposure. For example, the reconstituted glucose reagent kit is recommended to be stored in a

dark bottle, because on exposure to light a photochemical

reaction takes place and the reagent gets oxidised and

develops a pink color.

c. Color Instability

In some colorimetric reactions, the color may be stable for

only a short period of time. It is then necessary to time the

reaction carefully so that the readings of all samples and

standards are made during the time that the color remains

constant. Instability of color may be due to temperature

or absorption by the walls of the container where these

factors have an influence, they must be kept constant for

test samples and standards.

d. Foreign Matter and Air Bubbles

Solutions in the cuvettes must be free of lint or other

foreign matter, and air bubbles. A scrupulous cleaning

of the cuvettes and other glassware used in the analysis

should help to eliminate foreign matter from the solution.

For example, in a flow through cuvette of most semiautomated analyzers an air bubble trapped in it, will lead

to a decrease in optical density of the solution.

e. Errors of Weighing and Dilution

Simple errors of weighing and dilution in preparing

reagents, sample and standards can affect photometric

results appreciably. A good analytical balance and reliable

volumetric glassware should be used. For example, while

reconstituting a control serum care should be taken while

dispensing the volume of distilled water to the control

sera bulb. An error in dilution will result in change in the

concentration of the constituents.

Instrument

Instruments are capable of considerable precision of

measurement. The instrumental precision is of a higher

order than that normally resulting from the development

of color in the test solutions. Inherent errors of the solution

as mentioned above actually cause greater deviation than

do instrumental errors.

a. Light Source

Unless a double cell photometer is being used, the

consistency and reproducibility of the light source is

important. Fluctuations in voltage should be overcome by

the use of a voltage stabilizer in line-operated instruments.

Lamps should be allowed to warm up for at least 5 minutes

before steady output can be expected.

b. Stray Light

Stray light from windows or overhead lighting striking

the instrument can cause error since invisible particles

suspended in solutions can reflect these rays. The covering

of the cuvette compartment with a light-tight cover (as

usually provided with the instrument) before taking

readings will reduce this error.

c. Slit Width

In the prism spectrophotometer, the purity of the

monochromatic band depends on the width of the

entrance and exit slits. The use of a narrow slit width will

produce more accurate results.

d. Moisture

Moisture can be the cause of fluctuating readings in

spectrophotometric operation. In instruments employing

desicant, it is advisable to change the freshly dried silica

gel at regular intervals. This is particularly important in an

environment of high humidity.

e. Linearity of Photocell Response

Reliable results depend upon the current output of

the photocell being proportional to the light striking

the photocell. This relationship can be disturbed if the

photocell is not adequately protected from moisture and

from overheating. Some instruments are fitted with heat

absorbing filters in the optical system and provided with

thermal insulation of the light source.

Clinical Chemistry 465

f. Cuvettes

An important source of error and one which requires

constant checking is the cuvette. It is necessary that cuvettes

be optically matched, so that readings will not be influenced

by their individual variation when a series is used for making

measurements. When it is necessary to reuse cuvettes that

have not had adequate time to dry after cleaning, a rinse

with alcohol and ether or acetone may be used to speed up

the drying process. If cuvettes are dirty, etched, scratched

or marked with fingerprints, erroneous readings will result.

g. Wavelength Calibration

A wavelength scale which is off calibration can be a source

of error in spectrophotometers.

 

Filters are made of glass or dyed gelatine between glass

plates and have a limited transmission band at which they

transmit maximally. To understand the use of light filters

consider a bluish-green solution which absorbs light in the

red part of the spectrum. Such a solution when illuminated

by white light absorbs red color wavelengths and emits

bluish-green light together with a small amount of red.

The greater the concentration of the solution the smaller

the amount of red light transmitted. The most sensitive

readings of the galvanometer will therefore be obtained

by allowing only the transmitted red light to activate the

photoelectric cell. The red filter achieves this by stopping

the transmission of bluish-green light and allowing only

the red light to pass through the solution.

Clinical Chemistry 463

In the more expensive type of equipment, a diffraction

grating or a prism is used to obtain the required

wavelength.

In diffraction grating, the white light is dispersed

into a continuous spectrum. By turning a wavelength

adjustment, the grating is rotated and different parts of the

spectrum are allowed to fall onto the photocell.

In glass prism spectrophotometers, light is focused

onto the prism. Light passes through and forms an

extended spectrum. On adjusting the exit slit (wavelength

adjustment) light can pass through the cuvette, and

illuminate the photocell.

Cuvettes and Flow-through Cells

These are used to hold colored solutions and must be

scrupulously clean, with no dirty finger marks or spillage

of fluid on the outside of the optical side. Spillage of fluid

or dirty finger marks will absorb light and interfere in the

measurement of the color. Scratches on the glass must be

avoided and if badly scratched it must be discarded.

In order to speed up laboratory work, a more recent

development in colorimetry is the introduction of followthrough cells. These cells enable colorimetric readings to

be speeded up considerably, since the cells or cuvettes can

be drained without being removed from the colorimeters.

Photoelectric Cell

A photoelectric cell consists of photoelectric elements;

light falling on these elements generates an electric current

which deflects a galvanometer needle, the deflection being

proportional to the light intensity.

Galvanometer

The galvanometer measures the output of the

photosensitive element, and in good instruments a very

sensitive instrument is used.

Requirements of Colorimetric Analysis

When colorimetric determinations are made, it is essential

to ensure that the color being measured is only due to the

substance under investigation and is not due to any of

the reagents used. It is, therefore, essential to include the

following solutions.

1. Test solution

This contains the unknown concentration of the substance

together with the reagents used in the test.

2. Standard solution

This is usually identical to the test solution, except that

it contains a known amount of the substance being

determined and is approximately equal in concentration

to that expected in the test.

3. Blank solution

This solution is identical to both the test and standard

solution and it is carried through the complete test

procedure and contains all the reagents used, but

without any test or standard substance. Any color given

by the reagents used in the analysis can be detected and

eliminated.

In order to be sure that the absorbance is due solely to

the substance under test, the reading given by the ‘blank’

solution must be considered with the reading obtained

from the ‘test’ and ‘standard’ solutions. The photoelectric

absorptiometer is set to read zero absorbance with distilled

water. The blank, test and standard absorbance readings

are recorded, rechecking the zero absorbance between

each reading. The blank reading is then subtracted from

the test and standard reading as follows:

 Test – Blank _________________ × concentration of standard

Standard – Blank

This procedure will usually ensure that only the substance under investigation is being measured. Satisfactory

results are only obtained with absorbance ranging from

0.2 to 0.8, so that if possible the determination should

be modified in order that the lower and upper limits of

deflection fall within this range.

Sources of Error in Photometry

Errors in photometry can be attributed to three sources.

1. Inherent properties of the solution being measured

2. Instrument

3. Operator.

Inherent Properties of the Solution

The factors, which may be included in this group, influence

the absorption of light by the solution and can be the cause

of deviations from Beer’s Law.

a. Chemical Nature of the Solvent and Solution

Deviations from Beer’s law may occur either as a result

of a shift in the shape of a given portion of the absorption

curve as the concentration changes or because of the

absence of a linear relationship between optical density

and concentration. A shift in the shape of a portion of the

absorption curve can indicate a chemical transformation

of a portion of the colored component being analyzed into

a second component of a different color. The production

of a second colored component may also occur due to

an impurity in the solvent in which the original colored

464 Concise Book of Medical Laboratory Technology: Methods and Interpretations component is dissolved. For example, iodine dissolved in

carbon tetrachloride is deep purple but dissolved in alcohol

is brown. The presence of only 1% alcohol as an impurity

in carbon tetrachloride is sufficient to change the color and

hence, the shape of the absorption curve of iodine in carbon

tetrachloride. Thus, the absolute purity of the solvent is very

important in spectrophotometric work. This is particularly

true for analysis carried out in the ultraviolet region. The

breakdown of a linear relationship between optical density

and concentration can be due to the dissociation of a

colorless substance to give colored ions, or vice versa.

b. Exposure to Light

Certain compounds tend to bleach or discolor or get colored

when exposed to light. Such photochemical reactions are

likely to occur when the test sample is stored in a warm,

brightly-lighted room. This may occur while the sample

is in the photometer, if the intensity of illumination is too

high,