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2. The reason why decreased chloride levels often occur

in acute infections is not clear.

3. Chloride measurements are of limited value in

renal diseases for the reason that plasma chloride

can be maintained near normal limits even when a

considerable degree of renal failure is present.

4. Increased chloride levels occur in

 a. Cushing’s syndrome

 b. Dehydration

 c. Hyperventilation

 d. Eclampsia

 e. Anemia

 f. Cardiac decompensation

 g. Some renal disorders.

5. Decreased chloride levels occur in

 a. Severe vomiting

 b. Severe diarrhea

 c. Ulcerative colitis

 d. Pyloric obstruction

 e. Severe burns

 f. Heat exhaustion

 g. Diabetic acidosis

 h. Addison’s disease

 i. Fever

 j. Acute infections such as pneumonia

 k. Use of drugs such as mercurial and chlorothiazide

diuretics.

Interfering Factors

1. The plasma chloride concentration of infants is usually

higher than that of children and adults.

2. Many drugs may cause a change in chloride levels.

Be Careful

1. In intravenous therapy, if the solution contains

100 mEq/L, there is ample chloride present for the

correction of urine metabolic acidosis.

2. If an electrolyte disorder is suspected, daily weight and

accurate intake and output should be recorded.

SERUM IRON AND TIBC

Ferrozine Method

(Courtesy: Tulip Group of Companies)

For the determination of iron and total iron binding capacity

in serum (laboratory reagent for professional use only).

summary

Iron found in blood is mainly present in the hemoglobin

of the RBCs. Its role in the body is mainly in the

transportation of oxygen and cellular oxidation. Iron is

absorbed in the small intestine, and bound to a globulin

in the plasma, called transferrin and transported to

the bone marrow for the formation of hemoglobin.

Increased serum levels are found in hemolytic anemias,

hepatitis, lead and iron poisoning. Decreased serum

levels are found in anemias caused by iron deficiency

due to insufficient intake or absorption of iron, chronic

blood loss, late pregnancy and cancer. Increase in TIBC

is found in iron defecient anemias and pregnancy.

Decrease in TIBC is found in hypoproteinemia,

hemolytic/pernicious/sickle cell anemias, inflammatory

diseases and cirrhosis.

Principle

Iron, bound to transferrin, is released in an acidic medium

and the ferric ions are reduced to ferrous ions. The Fe (II)

ions react with ferrozine to form a violet colored complex.

Intensity of the complex formed is directly proportional to

the amount of iron present in the sample. For TIBC, the

serum is treated with excess of Fe (II) to saturate the iron

binding sites on transferrin. The excess Fe (II) is adsorbed

and precipitated and the Iron content in the supernatant is

measured to give the TIBC.

 Acidic Medium

Fe (III) Fe (II)

Fe (II) + Ferrozine Violet colored complex

Normal Reference Values

Serum Iron (Males) : 60–160 µg/dL

(Females) : 35–145 µg/dL

(Neonates) : 150–220 µg/dL

TIBC : 250–400 µg/dL

UIBC : 160–360 µg/dL.

It is recommended that each laboratory establish its

own normal range representing its patient population.

502 Concise Book of Medical Laboratory Technology: Methods and Interpretations Contents 35 mL 75 mL

Iron reagents

L1: Iron cutter reagent 35 mL 75 mL

L2: Iron color reagent 2 mL 4 mL

S: Iron standard (100 µg/dL) 2 mL 2 mL

TIBC reagents

L1: TIBC saturating reagent 10 mL 20 mL

L2: TIBC precipitating reagent 1 g 2 g

Storage/stability

Contents are stable at 2–8°C till the expiry mentioned on

the labels.

Reagent Preparation

Reagents are ready to use.

Sample Material

Serum, free from hemolysis. Iron is reported to be stable in

serum for 7 days at 2–8°C.

Procedure

Wavelength/filter : 570 nm (Hg 578 nm)/yellow

Temperature : RT

Light path : 1 cm

Iron Assay

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

standard (S), sample blank (SB) and test (T):

Addition

Sequence

B

(mL)

S

(mL)

SB

(mL)

T

(mL)

Iron buffer reagent (L1) 1.0 1.0 1.0 1.0

Distilled water 0.2 - - -

Iron standard (S) - 0.2 - -

Sample - - 0.2 0.2

Iron color reagent (L2) 0.05 0.05 - 0.05

Mix well and incubate at RT for 5 minutes. Measure the

absorbances of the blank (Abs B), standard (Abs S), sample

blank (Abs SB) and test sample (Abs T) against DW.

TIBC Assay

Pipette into a clean dry test tube

Serum

TIBC saturating reagent (L1)

0.5 mL

1.0 mL

Mix well and allow to stand at RT for 10 min and add

TIBC precipitating reagent (L2) Approx 50 mg

Mix well and allow to stand at RT for 10 minutes.

Centrifuge at 2500–3000 rpm for 10 minutes to obtain

a clear supernatant. Determine the iron content in the

supernatant as above mentioned iron assay.

Calculations

 Abs T- (Abs SB + Abs B) Iron in µg/dL = ________________________ × 100 Abs S – Abs B

 Abs T – (Abs SB + Abs B) TIBC in µg/dL = _______________________ × 300 Abs S – Abs B

UIBC in µg/dL = TIBC in µg/dL – Iron in µg/dL

Linearity

This procedure is linear upto 1000 µg/dL. If values exceed

this limit, dilute the sample with distilled water and repeat

the assay. Calculate the value using the proper dilution

factor.

Notes

Hemolysis interferes with the test as the hemoglobin

present in the RBCs has a very high iron content.

All glassware being used for the test should first be

rinsed with 1% or 0.1 N HCI and then with good quality

deionized water before use.

System Parameters

Reaction : End point + Interval : SB

Wavelength : 578 nm Sample

volume

: 0.2 mL

Zero setting : Deionized Reagent

volume

: 1.05 mL water

Incubation

temperature

: RT Standard : 100 ng/dL

Incubated

time

: 5 min Factor :

Delay time : — React slope : Increasing

Read time : — Linearity : 1000 µg/dL

No. of read : — Units : µg/dL

Clinical Relevance

1. TIBC is raised in

 a. Inadequate dietary iron

 b. Iron deficiency anemia due to hemorrhage

 c. Acute hepatitis

 d. Polycythemia

 e. Oral contraceptive use.

2. Decreased levels of TIBC are caused by

 a. Pernicious anemia

 b. Thalassemia

 c. Sickle cell anemia

Clinical Chemistry 503

 d. Chronic infection

 e. Cancer

 f. Hepatic disease

 g. Uremia

 h. Rheumatoid arthritis.

Interfering Factors

1. Transferrin is elevated in

 a. Children 2½ to 10 years of age

 b. Pregnant women during the third trimester

2. Drugs that may cause increased TIBC are

 a. Chloramphenicol

 b. Fluorides.

TRACE ELEMENTS

The term trace elements refers to inorganic substances

which occur in concentration < 0.01% of the body mass,

i.e. in amounts < 10–6 g/g of body weight. They are divided

into essential and nonessential trace elements. In humans,

Cr, Co, Cu, Fe, l, Mn, Mo, Ni, Se, Zn belong to the former

category; Al, Ag, As, Au, Ba, Bi, Cs, Cd, Pb, Ti, and V belong

to the group of nonessential trace elements. The latter also

include elements without physiological functions as well

as toxic heavy metals. Magnesium, in a strict sense, is not

a trace element but is customarily considered to be one. In

this issue three trace elements are considered.

ZINC

Oxidation state + 2,