Principle of Colorimetry in Medical Diagnostics
Colorimetry operates on the fundamental principle that substances absorb light at specific wavelengths, producing a measurable color change that correlates linearly with the concentration of the analyte being measured. 1
Core Mechanism
The colorimetric method measures the intensity of color produced when a substance reacts with specific reagents, based on the Beer-Lambert law which states that absorbance is directly proportional to concentration. 1 The technique involves:
- Light absorption at specific wavelengths - Different substances absorb light at characteristic wavelengths, creating distinct color patterns that can be quantified 1
- Linear relationship between concentration and absorbance - The intensity of the color produced is directly proportional to the concentration of the target substance, though this linearity can be lost at very high concentrations due to signal saturation 1
- Spectral analysis - The system measures how much light is absorbed versus transmitted through the sample at specific wavelengths 1
Technical Components
Light Source and Detection
The colorimeter uses specific wavelengths of light to optimize detection of target molecules. 1 For example:
- Hemoglobin detection utilizes wavelengths around 415 nm (blue) and 540 nm (green) where hemoglobin light absorption is optimized 1
- RGB signal processing converts red, green, and blue light signals into quantifiable data through mathematical transformation 1
Color Deconvolution
In multiplex assays, color deconvolution algorithms extract individual chromogen channels from composite RGB images, assuming a linear relationship between stain concentration and absorbance. 1 Each chromogen is assigned a unique color code for separate quantification. 1
Clinical Applications
Serum Albumin Measurement
The bromcresol green (BCG) colorimetric method is the most widely used clinical application, offering several advantages: 1
- Rapid and reproducible with automated processing capabilities 1
- Low coefficient of variation (5.9%) ensuring consistent results 1
- Small sample volume requirements with minimal interference from lipemia, salicylates, or bilirubin 1
- Normal range of 3.8-5.1 g/dL by BCG method, compared to 3.5-5.0 g/dL by electrophoretic methods 1, 2
Ketone Detection
The nitroprusside colorimetric reaction produces a purple color when acetoacetate reacts with sodium nitroferricyanide, enabling detection of ketosis. 1 This method is widely available in dipstick and tablet formats for both urine and blood testing. 1
Important Limitations and Pitfalls
Method-Specific Variations
Different colorimetric methods produce different results for the same sample: 1, 2
- BCG method overestimates albumin in the low range by approximately 0.61 g/dL and differs from electrophoretic methods by about 0.3 g/dL 1, 2
- BCP method underestimates serum albumin by approximately 0.71 g/dL in pediatric hemodialysis patients and by 19% compared to nephelometry 1, 2
- Always confirm which assay method your laboratory uses, as reference ranges vary significantly between methods 1, 2
Signal Saturation
After enzymatic chromogen signal saturation, the ratio of target to signal is no longer linear, compromising accuracy at very high concentrations. 1 This represents a critical limitation when measuring samples with elevated analyte levels. 1
Pre-analytical Factors
Prolonged tourniquet application during blood draw can cause hemoconcentration, artificially elevating protein measurements including albumin. 2 Laboratory error should always be ruled out with repeat testing before clinical interpretation. 2
Spectral Compensation Requirements
When multiple fluorochromes or chromogens are used simultaneously, spectral overlap must be corrected through compensation to prevent false-positive results. 1 This involves adjusting electronic circuits or using software algorithms to place populations in their respective fluorescence quadrants without overlap. 1