Principle of Measurement

1. Arsenazo III Method (Most widely used in modern analyzers)

How it works:

• Calcium ions react with Arsenazo III dye to form a blue-purple complex.

• The intensity of the color is measured photometrically at 650–660 nm.

• Highly specific for calcium with minimal interference from magnesium.

Advantages:

• Excellent stability

• Strong absorbance

• High specificity and precision

2. o-Cresolphthalein Complexone (OCPC) Method

How it works:

• Calcium forms a purple-red complex with o-cresolphthalein complexone in alkaline conditions.

• Read photometrically at 570–580 nm.

• Common in semi-auto chemistry analyzers.

Advantages:

• Reliable and widely established

• Suitable for manual and automated systems

Reagent Components

Arsenazo III Reagents May Contain:

• Arsenazo III dye

• Buffer (MES or other suitable buffers)

• Stabilizers

• Surfactants

• Preservatives

OCPC Reagents May Contain:

• o-Cresolphthalein complexone

• Alkaline buffer (e.g., 8-hydroxyquinoline to remove magnesium interference)

• Dye stabilizer

• Surfactants

• Preservatives

Packaging Format

• Single liquid reagent (most common)

• Two-reagent system (R1 + R2)

• Ready-to-use liquid stable bottles

• Analyzer-specific reagent cartridges

Common volumes: 25 ml, 50 ml, 100 ml, 250 ml or more.

Sample Types

• Serum

• Plasma (heparinized samples preferred)

• Urine (dilution may be needed)

Measurement Methods & Reagents

1. Ion-Selective Electrode (ISE) Method (Most common in modern analyzers)

Reagents include:

• Reference (internal standard) solution

• Electrode electrolyte solution

• Conditioning solution

• Cleaning solution

• Calibrators (Low and High Chloride)

Principle:

A chloride-selective membrane develops an electrical potential proportional to chloride concentration.
ISE systems require stable calibration solutions and maintenance reagents.

2. Mercuric Thiocyanate Colorimetric Method

How it works:

1. Chloride reacts with mercuric ions, forming mercuric chloride.

2. Released thiocyanate ions form a red-colored complex with ferric ions.

3. The color intensity is measured photometrically at 480–520 nm.

Reagents contain:

• Mercuric thiocyanate

• Ferric nitrate or ferric ammonium sulfate

• Buffer system

• Surfactants and stabilizers

Advantages:

• Reliable for automated chemistry analyzers

• High precision

3. Colorimetric TPTZ Method (Less common)

Chloride displaces iron from a complex; the released iron reacts with TPTZ dye to form a measurable color.

Packaging Formats

• Single-liquid reagent

• Two-reagent kits (R1 buffer + R2 color reagent)

• ISE-specific reagent packs

• Ready-to-use liquid stable formulations

Typical volumes: 25 ml, 50 ml, 100 ml, 250 ml.

Features / Components

Depending on the method (commonly direct enzymatic method), HDL reagent typically contains:

1. Detergents / Selective Chemicals

• These selectively mask or inactivate non-HDL lipoproteins (VLDL, LDL, chylomicrons), allowing accurate measurement of HDL only.

2. Enzymes

• Cholesterol esterase (CHE): Converts HDL-cholesterol esters into free cholesterol.

• Cholesterol oxidase (CHOD): Converts free cholesterol into cholest-4-en-3-one and hydrogen peroxide.

• Peroxidase (POD): Reacts with hydrogen peroxide to form a measurable colored compound.

3. Chromogenic Substrates

• Color-forming agents (e.g., 4-Aminoantipyrine) produce a detectable color reaction measured photometrically.

4. Buffers

• Maintain optimal pH for enzyme activity.

5. Stabilizers & Preservatives

• Ensure long shelf life and consistent performance.

🔬 HDL Chemistry Reagents (Clinical Chemistry / Lipid Panel)

HDL reagents are used to quantify High-Density Lipoprotein cholesterol in serum or plasma as part of a lipid profile. In semi-automated systems (bench-top analyzers, open systems, manual/semi-auto photometric readers), the reagents must be:

• Stable at 2–8°C

• Compatible with open-channel analyzers

• Ready-to-use or with simple reconstitution

• Optimized for endpoint or two-reagent homogeneous methods

1. Ion-Selective Electrode (ISE) Method

Most modern chemistry analyzers use ISE for sodium.

Reagents include:

• Reference solution / Internal standard

• Electrolyte solution for electrode stability

• Conditioning solution

• Cleaning solution

• Calibration solutions (Low & High Na⁺ standards)

Principle:

The sodium ISE membrane selectively binds Na⁺ ions, generating an electrical potential proportional to sodium concentration. Calibration solutions are crucial for accuracy.

2. Flame Photometry Method

Older or standalone electrolyte analyzers may use flame photometry.

Reagents include:

• Diluent

• Standard sodium solution

• Internal lithium standard (in some systems)

• Cleaning solutions

Principle:

Sodium emits light at a characteristic wavelength (589 nm) when introduced into a flame. The emitted intensity is proportional to concentration.

3. Colorimetric (Photometric) Method

Used in some semi-automatic chemistry analyzers when ISE is not available.

Reagents include:

• Ionophore-based chromogenic reagents

• Buffer solution

• Color-forming indicator dye

• Stabilizers and preservatives

Principle:

Sodium ions react with a specific ionophore dye to produce a measurable color change.

Reagent Format

Sodium reagent kits may include:

• Single bottle (for colorimetric systems)

• Multi-solution packs (ISE systems)

• Standards in sealed ampoules or bottles

• Ready-to-use liquid formulations

Sample Types

• Serum

• Plasma

• Urine (diluted appropriately)

• Whole blood (in ISE blood gas analyzers)

🔬 1. CHOD-PAP Method (Gold Standard)

Principle

1. Cholesterol esterase converts cholesterol esters → free cholesterol

2. Cholesterol oxidase converts cholesterol → cholest-4-en-3-one + H₂O₂

3. Peroxidase (POD) uses H₂O₂ to produce a colored dye

   • Common chromogens: 4-AAP, TOOS, DAOS, HDAOS

Absorbance: measured at 500–550 nm depending on chromogen.

What the Reagents Contain

R1 (Buffer / Chromogen)

• Good’s buffer (e.g., PIPES, TES)

• Phenol or TOOS/DAOS derivatives

• Stabilizers & surfactants

• Preservatives

R2 (Enzyme Reagent)

• Cholesterol esterase

• Cholesterol oxidase

• Peroxidase

• Additional surfactants