Oximetry measuring principle ABL800 FLEX Oximetry measuring principle RTC, December 2004 Radiometer Medical ApS, Åkandevej 21, DK-2700 Brønshøj, Tel: +45 38 27 38 27, www.radiometer.com

Agenda Principles of operation Technical aspects Calibration Absorption spectroscopy Measuring principle Technical aspects The hemolyzer The lamp unit Interfering substances Calibration Zero point Cuvette path length, tHb calibration Quality control Summary

Location

Oximetry parameters ctHb = concentration of total hemoglobin sO2 = oxygen saturation % FO2Hb = fraction of oxyhemoglobin FCOHb = fraction of carboxyhemoglobin FHHb = fraction of deoxyhemoglobin FMetHb = fraction of methemoglobin FHbF = fraction of fetal hemoglobin ctBil = concentration of total bilirubin

Diagram The optical system consists of a lamp unit, a hemolyzer containing a cuvette, an optical fiber and a spectrometer Optical fiber Hemolyzer Spectrometer Cuvette Sample in Lamp unit

Principles of operation Red blood cells contain hemoglobin Hemoglobins are colored proteins that absorb light Hemoglobin derivatives have different absorption spectra The concentrations of the various hemoglobin derivatives can be determined from the total absorption spectra Absorbance HHb O2Hb COHb MetHb 500 550 600 650 700 nm

White light White light is a mixture of light at different wavelengths Each wavelength corresponds to a specific color Prism White light

Absorption Blue Green Red Water When white light is sent through water, there is no absorption Light of all colors is transmitted, and light appears white Blue Green Red Water

Variation in concentration If a green substance is added to the water, the water looks green This is because the other colors are transmitted to a smaller degree If the concentration of the green substance is doubled, the sample appears “twice as” green since the solution absorbs even more blue and red light Water + 1 part green substance Blue Green Red Water + 2 parts green substance Blue Green Red

Variation in concentration Substances are characterized optically by individual absorption spectra - a kind of finger print The absorbance is proportional to the concentration 2 parts green substance Color/wavelength Absorbance Blue Green Red 1 part green substance

Video of absorption

A =  × c × l Lambert-Beer’s law Converting absorbance into concentrations Measured absorbance of a single substance is directly proportional to its concentration and the length of the light path through the sample A =  × c × l Where A = absorbance  = extinction coefficient c = concentration l = length of light path

When different components are present If two colored substances are mixed together, the light absorbed corresponds to a summation of each individual substance’s absorption A Absorbance Wavelength B Absorbance Wavelength A+B Absorbance Wavelength

l A ´ = c e Lambert-Beer’s law Measured absorbance of a single substance is directly proportional to its concentration l A y ´ = c e = absorbance of substance y at wavelength l = extinction coefficient of substance y at wavelength l = concentration of substance y in sample = length of light path Note that sometimes is given as a single constant

Absorption of blood The absorption spectrum of a blood sample is a summation of all the different hemoglobin spectra 500 550 600 650 700 nm Absorbance HHb O2Hb COHb MetHb SHb Bilirubin 1.5

Measuring principle A = log(Io/I) First the hemoglobins are released from the red blood cells by hemolyzation Light is transmitted through a cuvette containing the hemolyzed sample The spectrometer measures the intensity of the transmitted light Absorbance is derived as: A = log(Io/I) where Io is the reference intensity measured on calibration solution

Animation of oximetry measurement

Calculation F c O Hb tHb = The hemoglobin concentrations are determined by Lambert-Beer’s law The amount of light absorbed is related to the concentrations of the hemoglobin derivatives Absolute concentrations are not useful - hemoglobin derivatives are only useful in relation to tHb The fraction of the hemoglobin derivatives in relation to tHb is reported ctBil is given as concentration F c O Hb tHb 2 = Example:

The hemolyzer Contains the thermostatted (37 °C) cuvette Hemolyzation is the process of breaking down the cell walls to prevent scattering of the light releasing hemoglobins from inside the red blood cells This hemolyzation takes place inside the measuring cuvette by means of ultrasonic vibration under pressure prevents formation of air bubbles final results unbiased (especially O2Hb and sO2) Scattering of light leads to non-linear correlation between absorbance and concentration of hemoglobins

Animation of hemolyzation

The hemolyzer – ultrasonic self-cleaning Cleaning by ultrasonic vibration during every rinse The cleaning effectively removes any deposits in the cuvette No deposits  Higher accuracy

The hemolyzer – no clotting The hemolyzing unit acts as a clot trap Clots trapped in the hemolyzer will be destroyed during hemolyzation Before hemolyzation During hemolyzation No clotting + self-cleaning  NO maintenance

Lamp unit Halogen lamp Provides white light to the sample Photodiode Maintains constant intensity from halogen lamp Infrared filter Absorbs heat generated by infrared radiation Lens Focuses the light towards the cuvette Neon lamp Provides a reference light beam (spectral line) to the spectrometer

Reference light beam – neon lamp Built-in neon lamp provides a reference light beam to the spectrometer has one dominant wavelength, 585 nm monitor photodiode maintains the light intensity if there has been a drift in the wavelength measurement, the analyzer corrects accordingly Intensity photodiode Wavelength (nm)

ABL800 spectrometer The optical system is based on a 128-wavelength spectrometer with a measuring range of 478–672 nm The spectrometer consists of: a combined mirror and concave grating the grating separates the light into different wavelengths the mirror reflects and focuses the light onto the photodiode array a photodiode array, which contains 128 photodiodes that measure the intensity of light at 128 wavelengths Spectrometer Concave grating Photodiode array Slit

Why 128 wavelengths? More accurate spectrum A continuous spectrum Possible to more accurately detect interfering substances 500 550 600 650 700 nm Abs. HHb O2Hb COHb MetHb SHb Bilirubin 1.5 Bilirubin concentration in thís plot shows absorbance of 400 umol/L.

Getting the result The measured spectrum is compared to a model spectrum (“Best fit” method) The total amount of hemoglobin is reported, ctHb The amount of each hemoglobin derivative in the sample is reported as a fraction of ctHb Absorbance Wavelength Measured Model

The effect of interfering substances All colored substances which absorb light in the range of 476–672 nm will add to the measured spectrum = residual spectrum If the residual spectrum is registered too high, a warning is issued (“oxi spectrum mismatch”) Absorbance Wavelength Residual spectrum Measured Model

Dealing with interfering substances The following substances are always determined and therefore show no interference with the oxi results Intralipid Sulfhemoglobin Bilirubin Fetal hemoglobin is determined and, when above a certain value, is compensated for The following substances can in normal therapeutic concentrations cause minor interference (see reference manual) HiCN Evans Blue Methylene Blue

Calibration The optical system is calibrated by determining the zero point, Io, and the cuvette path length, L The zero point, Io is the intensity of light passing through a colorless solution The cuvette path length, L is the distance the light travels through the cuvette

Calibration Zero point is measured automatically at every 1-point and 2-point calibration The cuvette path length (length of light path, L) is determined from Lambert-Beer’s law by measuring the absorbance of the tHb Calibration solution A =  ×L × c = absorbance = extinction coefficient = concentration of optically active substance in tHb Calibration solution = length of light path L

tHb Calibration It is recommended that a tHb calibration is performed manually every 3 months

Summary Accuracy Reliability Maintenance free 128 wavelengths give a continuous spectrum No interference from frequently seen substances No deposits in the cuvette Reference neon lamp control of wavelength measurement Reliability No clotting No deposits Maintenance free Unique construction (cuvette and hemolyzer in one unit)

Radiometer Training Center, December 2004 Radiometer Medical ApS, Åkandevej 21, DK-2700 Brønshøj, Tel: +45 38 27 38 27, www.radiometer.com