Clinical Chemistry: Triglycerides and Chloride Analysis

Triglycerides

Constitute 95% of tissue storage fat and provide energy for the cell.

Clinical Significance of Triglycerides

High serum triglyceride levels are associated with important risks of atherosclerosis and can be due to diseases like lipid metabolism disorders, diabetes, renal or endocrine disorders.

Method (*Elitech)

In vitro diagnostic devices (reagent and standard) for professional use only, containing sodium azide which may react with lead or copper plumbing.

Warnings and Precautions for Triglycerides

Flush reagents with copious amounts of water to prevent azide buildup and adhere to good laboratory practice.

Stabilities

Store at 2 to 8°C, protect from light, and do not freeze or use after expiration dates.

Principle of Triglycerides Determination

Enzymatic determination of triglycerides according to specific reactions.

Reagents in Triglyceride Enzymatic Determination

Includes Reagent 1: R1, Reagent 2: R2, and Standard: Std**.

Calculation of Triglycerides Estimation

𝐴 𝑠𝑎𝑚𝑝𝑙𝑒 / 𝐴 𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑 × 𝑛.

Reference Values for Triglycerides

200 mg/dL, 2.28 mmol/L.

Chloride

Measured at pH 7.0 with a concentration of 50 mmol/L.

Warnings and Precautions for Chloride

Includes precautions for handling reagents and ensuring proper laboratory practices.

Expected Values for Chloride

Standard reference values as per laboratory guidelines.

Quality Control for Triglycerides

Ensures accuracy and reliability of test results.

Limitations of Triglycerides Testing

Factors that may affect the accuracy of triglyceride measurements.

Specimen Collection and Preparation

Guidelines for collecting and preparing samples for triglyceride testing.

Storage and Stability of Reagents

Instructions for proper storage conditions to maintain reagent efficacy.

Enzymatic-colorimetric Method

An end point method used for the determination of triglycerides.

Lipoprotein lipase

An enzyme with a reference value of ≥1,100 U/L.

Glycerol kinase

An enzyme with a reference value of ≥450 U/L.

Glycerol-3-phosphate oxidase

An enzyme with a reference value of ≥5,000 U/L.

ADPS

A reagent with a concentration of 0.9 mmol/L.

4-Aminoantipyrine (4-AAP)

A reagent with a concentration of 0.7 mmol/L.

Normal Level

<150 mg/dL (1.69 mmol/L)

Borderline high Level

150 - 199 mg/dL (1.69 - 2.25 mmol/L)

High Level

200 - 499 mg/dL (2.26 - 5.64 mmol/L)

Very High Level

≥ 500 mg/dL (5.65 mmol/L)

Fasting Requirement

Use serum or lithium heparinized plasma from fasting patients (≥12 hours)

Sample Limitations

Do not use icteric or hemolyzed samples; do not use other specimens

Usable Range Warning

Do not report results outside of the usable range

Unconjugated Bilirubin Interference

Negative bias from 9 mg/dL (90 mg/L, 154 μmol/L) on normal serum

Conjugated Bilirubin Interference

Negative bias from 6.2 mg/dL (62 mg/L, 107 μmol/L) on normal serum

Haemoglobin Interference

No significant interference up to 500 mg/dL (5 g/L) on normal serum

Ascorbic Acid Interference

No significant interference up to 2 mg/dL (20 mg/L, 0.11 mmol/L)

Glucose Interference

No significant interference up to 500 mg/dL (5 g/L, 28 mmol/L)

Manual Procedure Wavelength

Wavelength: 550 nm

Manual Procedure Optical Path

Optical path: 1 cm

Sample/Reagent Ratio

Sample/reagent ratio: 1:100

Incubation Temperature

Temperature: 37°C

Reagent R Volume

Reagent R: 300 μL

Standard/Calibrator Volume

Standard/Calibrator: 3 μL

Sample Volume

Sample: 3 μL

Incubation Time

Incubate at 37°C for 8 minutes

Calculation of Triglycerides Estimation

Formula 1. Calculation of Triglycerides Estimation.

Sample Storage Stability

Samples are stable 5 to 7 days if stored at 2 to 8°C, 3 months at -15 to -20°C and several years at -70°C

False Results Warning

Sampling could lead to false results if performed during or immediately after the administration of some drugs

n

standard concentration

mg/dl x 0.0113

conversion to mmol/L

mg/dl x 0.01

conversion to g/L

Classical methods

based on the combination of chloride ion with either silver or mercury to form the undissociated chloride compound

Titrimetric technique of Shales and Shales 1

most popular method for the estimation of chloride in body fluids since 1941

Standardized solution of mercuric nitrate

used in the titrimetric technique along with diphenyl carbazone as an indicator

Colorimetric procedure

based on the water analysis scheme of Zall et al. and later adapted to automation by Skeggs and Hochstrasser

Chloride reaction with mercuric thiocyanate

releases thiocyanate ions which combine with ferric ions to form reddish ferric thiocyanate

Absorbance measurement

of the stable-colored compound at 500 nm compared to that of a chloride standard

Actual Procedure in the Laboratory

1000 μL of Reagent + 10 μL of Serum, incubated at room temperature for 15 minutes before reading through humalyzer

Color change during procedure

changes from colorless to yellowish (serum) to reddish

Ferric thiocyanate formation

chloride ions lead to this formation in the presence of ferric nitrate and mercuric thiocyanate

Coloration intensity

is proportional to the chloride concentration

Serum normal range

98 to 107 mEq/L

CSF normal range

118 to 132 mEq/L

Urine normal range

110 to 250 mEq/24h, varies with diet

Direct Chloride Color Reagent

Cat. No. 0211, contains solution of Mercuric Thiocyanate, 1.0 mmol/L, Ferric Nitrate, 37.5 mmol/L, Mercuric Nitrate, 0.155 mmol/L in methanol and water, and also contains nitric acid and surfactants

Direct Chloride Standard

100 meg/L, Cat.

Aqueous solution of Sodium Chloride

A solution containing sodium chloride dissolved in water.

ELITROL I

Normal control serum recommended for quality control.

ELITROL II

Abnormal control serum recommended for quality control.

Direct Chloride Color Reagent

A toxic and caustic product used in chloride testing.

Beer's Law

A principle that does not apply to the chloride reaction.

Linear range

The range of 70 to 120 mEq/L for the chloride reaction.

Calibration curve

A graph plotting the concentration of standards versus absorbance readings.

Chloride stability

Chloride in serum, plasma, urine, and spinal fluid is stable for at least 1 week at refrigerator or room temperature.

Dilution factor for urine

Results must be multiplied by a dilution factor of 3 when diluted with deionized water (1 part urine + 2 parts water).

Triglycerides synthesis

Triglycerides are synthesized in the kidney and intestine.

Enzymatic-colorimetric method

The method used for triglyceride determination.

Specimens for triglycerides

Serum and Lithium Heparinized Plasma can be used for triglyceride testing.

Wavelength for analysis

The wavelength used is 500 nm.

Temperature for analysis

The temperature used is 30°C.

Volume of reagent and sample

1000 μL Reagent, 10 μL Sample is used in the actual experiment for triglycerides.

Ferric thiocyanate complex

Characterized by a yellow-colored solution, with coloration intensity proportional to chloride concentration.

Ions released after chloride reaction

Thiocyanate Ions are released after the reaction of chloride with mercuric thiocyanate.

Volumes in calibration

300 μL of Reagent R + 300 μL of Standard/Calibrator in the CALIBRATION.

Quality control expected values

Normal chloride levels in CSF range from 98-107 meq/L.

Chloride reaction and Beer's Law

The chloride reaction does not follow Beer's Law but has a linear range of 70 to 120 mEq/L.

Verification of linear range

The linear range of an instrument can be verified before the actual testing.