NPN LAB (Ammonia)

1. Two-step approach
2. One-step approach

2 Approaches for Plasma Ammonia

Two-step approach

Approaches for Plasma Ammonia

> Ammonia is isolated from the sample ---> assayed

One-step approach

Ammonia is DIRECTLY measured on the sample using an enzymatic method or ion-selective electrode

1. Enzymatic Method
2. Dry slide method
3. Berthelot Reaction '
4. Nessler's Reaction

Laboratory Methodologies & Analytical Techniques: AMMONIA

Glutamate Dehydrogenase

Enzymatic Method of AMMONIA

340 nm

(ENZYMATIC METHOD OF AMMONIA) Glutamate Dehydrogenase - measure decrease in absorbance at ____ nm

Dry slide method

Alkaline buffer is employed to convert ammonium ions to ammonia gas with bromphenol blue as an indicator

buffer: Alkaline buffer

converts: Ammonium ion to Ammonia gas

indicator: bromphenol blue

Dry slide method

buffer:

converts:

indicator:

Dry slide technology uses spectrophotometer

TAKE NOTE!

1. Spreading Layer
2. Scavenger Layer
3. Reagent Layer
4. Indicator Layer
5. Support layer

Dry Slide Method layers

Spreading Layer

Dry Slide Method layers

> samples are distributed evenly

Scavenger Layer

Dry Slide Method layers

> filters out substances that interfere with the results (optional layer)

Reagent Layer

Dry Slide Method layers

> Reagent reacts with the sample

Indicator Layer

Dry Slide Method layers

> Reacted sample collects for spectral analysis

Support layer

Dry Slide Method layers

> Optical interface

> ARTERIAL blood is the preferred specimen

> Since ammonia is UNSTABLE, specimens must be kept at 4°C immediately after collection and transported on ICE

> VENOUS blood is not recommended, but if used, should be obtained without trauma, tourniquets should be used minimally, and fist clenching and relaxing avoided during collection.

> HEMOLYSIS should be avoided

PRECAUTIONS & SPECIMEN CONSIDERATIONS: AMMONIA

> ____ blood is the preferred specimen

> Since ammonia is ____, specimens must be kept at ___ °C immediately after collection and transported on ____

> _____blood is not recommended, but if used, should be obtained without trauma, tourniquets should be used minimally, and fist clenching and relaxing avoided during collection.

> ____should be avoided

Centrifugation: 0 to 4°C within 20 minutes of collection and immediately separated

Assayed: ASAP or frozen (-20degreeC)

Avoid: smoking several hours before ammonia testing

PRECAUTIONS & SPECIMEN CONSIDERATIONS: AMMONIA

Centrifugation:

Assayed:

Avoid:

Tobacco smoke

This can cause ammonia contamination

U- urine
A- ammonia in detergents
G- glassware
R- reagents
W- water

¤ Other sources of contamination of AMMONIA:

U
A
G
R
W

> Diffusion method: 20-120 μg/dL (12-70 μmol/L)

> Enzymatic method: 40-80 μg/dL (23-47 μmol/L)

> Resin method: 12-48 μg/dL (7-28 μmol/L)

Reference Interval / Range : AMMONIA

> Diffusion method:

> Enzymatic method:

> Resin method: