1. Oximetry measuring principle
ABL800 FLEX
Oximetry measuring principle
RTC, December 2004
Radiometer Medical ApS, Åkandevej 21, DK-2700 Brønshøj, Tel: ,

2. 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

3. Location

4. 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

5. 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

6. 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

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

8. 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

9. 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

10. 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

11. Video of absorption

12. 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

13. 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

14. 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

15. 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

16. 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

17. Animation of oximetry measurement

18. 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:

19. 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

20. Animation of hemolyzation

21. 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

22. 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

23. 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

24. 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)

25. 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

26. 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.

27. 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

28. 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

29. 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

30. 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

31. 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

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

33. 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)

34. Radiometer Training Center, December 2004
Radiometer Medical ApS, Åkandevej 21, DK-2700 Brønshøj, Tel: ,