Spectrophotometry & Quality Control

What is the theory of spectrophotometry as it applies to the clinical lab

Due to a molecule's structure, it may have the property of absorbing light of a certain wavelength. The amount or intensity (absorbance) of the molecule varies with the concentration of the substance

Most analytes in blood do not absorb light, so what is the solution?

A reagent is added to the blood to create a chromophore that will absorb light at a specific wavelength. The chromophore absorbance is related to the analyte concentration

What does a spectrophotometer measure?

Measures the amount of photons absorbed once a light is passed through a sample solution

Most labs utilize _____, which provides the largest separation of colors and has extreme accuracy

Diffraction Grating

What is the prism component of a Spectrophotometer

Monochromator

What is the graphical relationship between absorbance and transmittence?

Indirectly proportional

What is an absorbance spectrum?

A graph of wavelength (x) and absorbance (y) that will show the wavelength of maximum absorption, which will be the desired wavelength for the assay (as it will be most sensitive at this wavelength)

What is Beer's Law?

The concentration of a substance is directly proportional to the amount of light absorbed... A=ebc

What is the equation that related A to %T?

A=2-log(%T)

What is a standard (calibration) curve?

Known concentrations of standards are plotted for their absorbance, where then one can use unknown concentrations on the same graph to determine the concentrations based on the absorbance provided from the spectrophotometer

How can one use the absorption & concentration of standards to determine those measurements of unknown samples?

(Absorption of unknown / Absorption of standard) = (concentration of unknown / Concentration of standard)

What is the difference between a sample and reagent blank?

Both are subtracting interference (sample and reagent, respectively)

What is the language used when there are graphical deviations from Beer's law? What may cause these deviations?

Out of linearity. High concentrations and stray light.

What is nephelometry?

Measures scattered light from AB/AG complexes. Often at a 90 degree angle, and amount of scatter is directly proportional to amount of antigen concentration (antibody should be in excess)

What is flourometry?

Detects fluorescence (which occurs when a molecule absorbs light at one wavelength and re-emits it at a longer wavelength). Must be done at 90 degrees.

What is turbidimetry?

Measures absorbance (light blocked) by insoluble complexes. More absorbance = less light blocked = fewer complexes

What is luminometry?

Using oxidation reactions that give off a burst of light to measure the concentration of an analyte (larger burst = more analyte)

What is oximetry?

A solution is scanned for multiple wavelengths (commonly performed during BLOOD GAS analyses)

Lab statistics and reference ranges are usually based on WHAT kind of distribution?

Guassian (normal)

What is the confidence interval for most labs?

95%

What is analytical sensitivity?

Ability of a lab test to detect target analyte (limit of detection)

What is analytical specificity?

Ability of a lab test to detect intended analyte

What is clinical sensitivity?

The assay's ability to correctly (POSITIVELY) diagnose patients with disease (TP/(TP+FN))

What is clinical specificity?

The assay's ability to correctly identify a patient without the disease as negative (TN/(TN+FP))

What is a control vs a standard?

Controls are unknown, though they are often given as normal or abnormal controls. Standards have known concentrations, often used for calibration

What are delta checks?

Patient results compared to previously determined value on the patient (measured daily, typically stable value)

In a Levey-Jennings chart, what does it mean for it to be "in control"

8-9 points between 2 SD limits

In a Levey-Jennings chart, what is a shift?

Abrupt change in pattern

In a Levey-Jennings chart, what is a trend?

gradual change in pattern

Westgard Multi rules: 1 (2S) (warning or rejection?)

1 control exceeds mean / crosses +/- 2SD. WARNING

Westgard Multi rules: 1 (3S) (warning or rejection? random or systemic?)

1 control observation exceeds mean / crosses +/- 3SD. Rejection. Random.

Westgard Multi rules: R (4S) (warning or rejection?)

1 observation exceeds mean +2SD and the other exceeds the mean -2SD. Rejection. Random.

Westgard Multi rules: 2 (2S) (warning or rejection? random or systemic?)

2 consecutive observations exceeding mean +/- 2SD. Rejection. Systemic.

Westgard Multi rules: 4 (1S) (warning or rejection? random or systemic?)

4 consecutive observations exceeding same mean +/- 1 SD. Rejection. Systemic.

Westgard Multi rules: 10x (warning or rejection? random or systemic?)

10 consecutive observations falling on one side of the, above or below. Rejection. Systemic.

What is accuracy?

agreement between measured amount and true value

What is precision?

how reproducible measurements are

Where do the most errors occur in the pre/analytical/post system?

Preanalytical (specimen collection, transport)

What is internal QC vs external QC?

Internal (daily runs, establishing reference ranges), External (CAP, CLIA, Joint Commission)