Principle

Phosphate ions in an acidic medium react with ammonium

molybdate to form a phosphomolybdate complex. This

complex reacts with metol and is reduced to a molybdinum

blue complex. Intensity of the molybdinum blue complex

formed is directly proportional to the amount of inorganic

phosphorus present in the sample.

Phosphorus + Ammonium Molybdate→Phosphomolybdate complex

Phosphomolybdate complex + Metol→Molybdinum Blue Complex

Normal Reference Values

Serum (Adults ) : 2.5–5.0 mg/dL

(Children) : 4.0–6.5 mg/dL

Urine : 0.3–1.0 g/24 h

It is recommended that each laboratory establish its

own normal range representing its patient population.

Contents 10 Tests 25 Tests

L1: Acid reagent 30 mL 75 mL

L2: Molybdate reagent 30 mL 75 mL

L3: Color reagent 30 mL 75 mL

S: Phosphorus standard (5 mg/dL) 5 mL 5 mL

Storage/Stability

Reagents are stable at RT (25–30°C) till the expiry

mentioned on the labels.

Reagent Preparation

Reagents are ready to use.

Sample Material

Serum, heparinized/EDTA plasma or urine. Acidify the

urine with a few drops of cone. Hydrochloric acid and

dilute 1 + 19 before the assay, (results x 20). Inorganic

phosphorus is reported to be stable in serum for 7 days at

2–8°C.

Procedure

Wavelength/filter : 650 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)

Acid reagent 1.0 1.0 1.0

Molybdate reagent 1.0 1.0 1.0

Distilled water 0.1

Phosphorus standard(s) - 0.1

Sample - - 0.1

Color reagent 1.0 1.0 1.0

Clinical Chemistry 499

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

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

T) against the blank, within 30 minutes.

Calculations

 Abs T

Phosphorus in mg/dL = ________ × L5 Abs S

Linearity

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

this limit, dilute the sample with distilled water and repeat

the assay. Multiply the value obtained with an appropriate

dilution factor.

Notes

Hemolysis interferes with the test.

Use clean glassware washed with N/10 HCI as many

detergents contain phosphate ions.

The addition sequence of the reagents and the sample

is important and should not be changed.

System Parameters

Reaction : End point Interval :

Wavelength : 650 nm Sample

volume

: 0.1 mL

Zero setting : Reagent blank Reagent

volume

: 3.00 mL

Incubation

temperature

: RT Standard : 5 mg/dL

Incubated

time

: 5 min Factor :

Delay time : — React slope : Increasing

Read time : — Linearity : 15 mg/dL

No. of read : — Units : mg/dL

Normal Values

SI units

Adults < age 60 2.7–4.5 mg/dL 0.87–1.45 mmol/L

Females > age 60 2.8–4.1 mg/dL 0.90–1.30 mmol/L

Males > age 60 2.3–3.7 mg/dL 0.74–1.20 mmol/L

Cord blood 3.7–8.1 mg/dL 1.20–62 mmol/L

Premature infant 5.4–10.9 mg/dL 1.74–3.52 mmol/L

Newborn 4.5–9 mg/dL 1.45–2.91 mmol/L

Infant

(10 days–24 months) 4.5–6.7 mg/dL 1.45–2.16 mmol/L

Child

(24 months–12 years) 4.5–5.5 mg/dL 1.45–1.78 mmol/L

Clinical Relevance

Hyperphosphatemia (Increased Phosphorus Levels)

The most common causes of elevated blood phosphate

levels are found in association with kidney dysfunction and

uremia. This is because phosphate is so closely regulated

by the kidneys.

Increased phosphorus levels are associated with

a. Renal insufficiency and severe nephritis accompanied

by elevated BUN and creatinine.

b. Hypoparathyroidism (accompanied by elevated

phosphorus, decreased calcium, and normal renal

function).

c. Hypocalcemia

d. Excessive intake of alkali (possible history of peptic

ulcer)

e. Excessive intake of vitamin D

f. Fractures in the healing stage

g. Bone tumors

h. Addison’s disease

i. Acromegaly.

Hypophosphatemia (Decreased Phosphorus Levels)

Decreased phosphorus levels may be associated with

a. Hyperparathyroidism (accompanied by increased

calcium, no renal disease)

b. Rickets (childhood), osteomalacia (adults)

c. Diabetic coma because of increased carbohydrate

metabolism

d. Hyperinsulinism

e. Continuous administration of intravenous glucose in

a non-diabetic patient.

Interfering Factors

1. Normally high in children

2. Falsely increased by hemolysis of blood

3. Drugs causing possible elevation

 a. Diphenylhydantoin (phenytoin)

 b. Heparin

 c. Pituitrin

 d. Vitamin D

 e. Methicillin

 f. Tetracyclines

 g. Alkaline antacids

 h. Lipomol.

4. The use of laxatives or enemas containing large

amounts of sodium phosphate will cause increased

phosphorus levels.

5. Drugs causing possible decreases

 a. Aluminum hydroxide

 b. Epinephrine (adrenaline)

 c. Insulin

500 Concise Book of Medical Laboratory Technology: Methods and Interpretations  d. Mannitol

 e. Mithramycin

 f. Parathyroid injection.

CHLORIDE

Thiocyanate Method

(Courtesy: Tulip Group of Companies)

Chloride is a major extracellular anion and maintains the

cation/anion balance between intra and extracellular fluids,

mostly as a salt with sodium. Increased levels are usually

found in dehydration, kidney dysfunction, and anemia.

Decreased levels are found in extensive burns, vomiting,

diarrhea, intestinal obstructions, and salt losing nephritis.

Principle

Chloride ions combine with free mercury ions and release

thiocyanate from mercuric thiocyanate. The thiocyanate

released combines with the ferric ions to form a red brown

ferric thiocyanate complex. Intensity of the color formed is

directly proportional to the amount of chloride present in

the sample.

2 Cl– + Hg (SCN)2 HgCI2 + 2 (SCN)–

3 (SCN) + Fe3+ Fe(SCN)3

Normal Reference Values

Serum/plasma chloride : 96–106 mmol/L

Urine chloride : 170–250 mmol/24 h

CSF chloride : 120–135 mmol/L

It is recommended that each laboratory establish its

own normal range representing its patient population.

Chloride Kit

L1 : Chloride reagent 75 mL

S : Chloride standard (100 mmol/L) 5 mL

Storage/Stability

All reagents are stable at RT till the expiry mentioned.

Reagent Preparation

Reagents are ready to use.

For chloride: Serum, plasma, urine, and CSF. Dilute urine

samples 1 + 1 with distilled water before the assay. Chloride

is reported to be stable in serum for 7 days at 2–8°C.

Procedure

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

Temperature : RT

Light path : 1 cm

Chloride Assay

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

standard (S), and test (T).

Addition

Sequence

B

(mL)

S

(mL)

T

(mL)

Chloride reagent (L1) 1.0 1.0 1.0

Deionised water 0.01 – –

Chloride standard (s) – 0.01

Sample – – 0.01

Mix well and incubate at RT for 2 minutes. Measure the

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

T) against blank, within 60 minutes.

Linearity

The Chloride assay is linear between 70–140 mmol/L. If

values exceed this limit, dilute the sample with deionized

water (free from Na+/K+/Cl–

 ions) and repeat the assay.

Calculate the value using the proper dilution factor.

Notes

Bring all reagents to RT before use.

Turbid or icteric samples may produce falsely elevated

results.

The procedure for chloride measures total halides

such as bromides, iodides, and fluorides in addition to

chlorides hence, their contamination should be avoided.

Since the test is temperature sensitive, so a constant

temperature should be maintained during incubation

and reading.

System Parameters, Cl–

Reaction : End point Interval :

Wavelength : 505 nm Sample

volume

: 0.01 mL

Zero setting :  Reagent

blank

Reagent

volume

: 1.00 mL

Incubation

temperature

: RT Standard : 100 mmol/L

Incubated time : 2 min Factor :

Delay time : — React slope : Increasing

Read time : — Linearity :  70–140

mmol/L

No. of read : — Units : mmol/L

Calculation

 Abs. T

Chloride in mmol/L = _________ × 100 Abs. S

Clinical Chemistry 501

Normal Values

SI units

Children and adults 97–106 mEq/L 97–107 mmol/L

Premature infants 95–110 mEq/L 95–110 mmol/L

Full-term infants 96–106 mEq/L 96–106 mmol/L

Panic levels < 80 mEq/L < 80 mmol/L

> 115 mEq/L > 115 mmol/L

Clinical Relevance

1. Whenever, the serum level is much lower than 100

mEq/L, the urinary excretion of chloride falls to a very

low level.