🔬 1. IFCC Kinetic ALP Method (Standard)

Principle

ALP + p-Nitrophenyl phosphate (pNPP)
→ p-Nitrophenol (yellow) + Phosphate

• Absorbance increases as p-nitrophenol is formed.

• The rate of increase (ΔA/min) at 405 nm is proportional to ALP activity.

Measurement

• Wavelength: 405 nm

• Mode: Kinetic (multiple interval readings)

• Temperature: 37°C (IFCC standard)

🧪 2. Reagent Composition (Typical)

Most ALP kits use single-reagent or two-reagent formats.

R1 (Main Buffer Reagent)

• Diethanolamine (DEA) buffer (high pH ~10.3)

• Magnesium ions (Mg²⁺) — ALP cofactor

• Zinc ions (Zn²⁺) — enzyme stabilizer

• Surfactants

• Preservatives

R2 (Substrate Reagent)

• p-Nitrophenyl phosphate (pNPP)

• Stabilizers

• Preservatives

🔬 1. IFCC Kinetic Method (Standard AST Method)

Primary Reaction (AST enzyme reaction)

L-Aspartate + α-Ketoglutarate
→ Oxaloacetate + L-Glutamate

Indicator (Coupled) Reaction

Oxaloacetate + NADH + H⁺
→ Malate + NAD⁺
(catalyzed by MDH)

LDH is often included to convert pyruvate → lactate, preventing inhibition.

As AST activity increases, NADH decreases, causing a fall in absorbance at 340 nm.

Measurement

• Wavelength: 340 nm

• Mode: Kinetic (rate), ΔA/min

• Rate proportional to AST activity (U/L)

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.

1. Jaffe Method (Most Common)

• Based on the reaction between creatinine and picric acid in an alkaline medium.

• Produces an orange-red complex.

• Absorbance is measured photometrically, usually at 505–520 nm.

• Some kits include compensated Jaffe formulations to reduce interference from glucose, ketones, and proteins.

2. Enzymatic Method

• Uses a sequence of enzyme reactions (creatininase, creatinase, sarcosine oxidase, peroxidase).

• Produces a colored dye measurable at 550–570 nm.

• Higher specificity than Jaffe, minimal interference.

Reagent Components

Depending on the method, creatinine reagent kits may include:

For Jaffe Method

• Alkaline buffer

• Picric acid

• Stabilizers

• Surfactants

• Preservatives

For Enzymatic Method

• Enzyme mixture (creatininase, creatinase, sarcosine oxidase)

• Chromogenic dye reagents

• Phosphate or Good’s buffer

• Activators

• Preservatives

Packaging Format

Most creatinine reagent kits are provided as:

• Two-reagent system (R1 buffer + R2 working reagent)

• Ready-to-use liquid formulations

• Optional standards or calibrators

Common sizes: 25 ml, 50 ml, 100 ml, 250 ml, or analyzer-specific cartridges.

Sample Type

• Serum

• Plasma (heparin, EDTA)

• Urine (often diluted before analysis)

🔬 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

Principle of Measurement

1. UV Phosphomolybdate Method (Most modern analyzers)

How it works:

1. Inorganic phosphate reacts with ammonium molybdate in an acidic medium.

2. Forms a phosphomolybdate complex.

3. The complex is measured photometrically in the UV range (usually 340 nm).

4. The absorbance is directly proportional to phosphate concentration.

Advantages:

• Fast and highly specific

• Suitable for fully automated analyzers

• Minimal interference

2. Reduced Phosphomolybdate Method (Colorimetric)

How it works:

1. Phosphomolybdate complex is formed.

2. The complex is reduced to a blue-colored compound (“molybdenum blue”).

3. Measured at 600–700 nm, depending on dye.

Advantages:

• Strong color intensity

• Stable reaction for manual or semi-automatic systems

Reagent Components

Phosphorus reagent kits typically include:

R1 (Acid/Molybdate reagent)

• Ammonium molybdate

• Sulfuric acid or perchloric acid

• Stabilizers

• Surfactants

• Preservatives

R2 (Reducing reagent) (if applicable)

• Reducing agents (e.g., ascorbic acid, stannous chloride)

• Dye stabilizers

Some kits are single-reagent formulations.

Packaging Formats

• Liquid-stable ready-to-use reagents

• Two-reagent systems (R1 + R2)

• Analyzer-specific cartridges

• Optional calibrators and controls

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