1. 1 Radiometer Medical ApS, Åkandevej 21, DK-2700 Brønshøj, Tel: +45 38 27 38 27, www.radiometer.com RTC, December 2004 ABL800 FLEX Calibration

2. 2 Agenda What is calibration? Calibration material Why calibrate? Obtaining a calibration line Sensitivity Status Drift pO 2 electrode Metabolite electrodes Calibration results

3. 3 What is calibration? Liquid or gas with a known parameter value is drawn into the measuring chamber The electrode measures an electrical signal corresponding to the known value of the liquid or gas This is repeated with liquid or gas of another known parameter value The analyzer establishes a calibration line for each parameter and adjusts its readings to measure the known value of each parameter “The calibration process determines and checks the accuracy with which the ABL measures its parameters” (quote from the Operator’s Manual)

4. 4 Calibration material Two calibrating solutions, Cal 1 and Cal 2, are used to calibrate the pH, electrolyte and metabolite electrodes Two gas mixtures, Gas 1 and Gas 2, are used to calibrate the pCO 2 and pO 2 electrodes The tHb Calibration Solution is used to calibrate the optical system

5. 5 Calibration solutions and gases Composition: Cal 1 contains: K +, Na +, Ca 2+, Cl -, Glu, Lac, pH = 7.4 Cal 2 contains: K +, Na +, Ca 2+, Cl –, pH = 6.8 Gas 1 contains: O 2 (19.7 %), CO 2 (5.6 %) Gas 2 contains: CO 2 (11.2 %) Nominal concentrations/percentages stated on container label Precise concentrations/percentages contained in the bar code

6. 6 Why calibrate? The electrodes are active elements and must be calibrated regularly Signals from the electrodes change because of, e.g. ­ protein build-up ­ worn-out membranes ­ aging electrodes

7. 7 Calibration line The calibration line represents the relationship between the electrical signal measured at the electrode and the concentration of the substrate specific to the electrode 6.800 7.400 –106 –69 pH mV 7.4006.800

8. 8 Obtaining a calibration line Each electrode has a different calibration line ­ the pH electrode is used as an example The electrical signals from two calibration solutions of known pH are plotted ­ Cal 1 solution with pH = 7.400 gives a reading of –106 mV ­ Cal 2 solution with pH = 6.800 gives a reading of –69 mV 6.800 7.400 –106 –69 mV pH

9. 9 Obtaining a calibration line The relationship between measured potential and concentration is linear The calibration line forms the scale used to convert the measured potential to pH 6.800 7.400 –106 –69 mV pH

10. 10 A 1-point calibration line versus a 2-point Only Cal 1 is measured, giving one calibration point A calibration line with the same slope as the previous 2-point calibration is superimposed on this new calibration point –106 –69 mV pH 6.8007.400 2P cal.

11. 11 Monitoring the calibration line The calibration line is updated at every calibration and is monitored with respect to: Sensitivity Status/zero point Describes the slope and position of the calibration line Drift Describes the variation in the calibration line between consecutive calibrations Each electrode has its own status and sensitivity limits

12. 12 Sensitivity (slope) The sensitivity is an evaluation of the slope of a 2-point calibration line in relation to the slope of the theoretical calibration line ­ the theoretical calibration line is derived from the Nernst equation The theoretical pH calibration line has a slope = –61.5 mV/pH 6.800 7.400 –106 –69 mV pH Actual Theoretical

13. 13 Sensitivity, an example During a 2-point calibration the following is measured: 7.400 –106 –69 mV pH –101 –65 EE  pH Theo. 2P-cal.  E –101.0 – (–65.0)  E –101.0 – (–65.0)  pH × (–61.5) (7.4–6.8)×(–61.5)  pH × (–61.5) (7.4–6.8)×(–61.5) Sensitivity = × 100 % Example: Sensitivity = × 100 % = 97.6 % 6.800 - Cal 1 solution with pH = 7.400 gives a reading of –101.0 mV - Cal 2 solution with pH = 6.800 gives a reading of –65.0 mV

14. 14 ABL800 FLEX sensitivity limits EE  pH Theo. 2P-cal. EE  pH Theo. 2P-cal. % ParameterSensitivity limits pH92-103 % pCO 2 84-100 % pO2pO2 5-40 pA/mmHg (37.5-300 pA/kPa) cK+cK+ 92-105 % cNa + 90-105 % cCl - 85-105 % cCa 2+ 90-105 % cGlu100-1800 pA/mM cLac150-2000 pA/mM For a calibration to be accepted, the sensitivity must fall within the following limits:

15. 15 Status The status describes the deviation of a 1-point calibration point from the theoretical calibration line ­ the status is evaluated using only one calibration point; i.e. we assume that the sensitivity (slope) is unchanged  E –100 – (–106) –61.5–61.5 Status = 7.40 +Example: Status = 7.40 + = 7.30 6.800 7.400 pH –69 Theo. 1P-cal. mV –106 –100 EE 7.300

16. 16 ABL800 FLEX status limits For a calibration to be accepted, the status and zero point must fall within the following limits Theo. cal. Theo. cal. % ParameterStatus limits pH6.7-8.1 pCO 2 6.2-260 mmHg cK+cK+ 0.5-12 mmol/L cNa + 10-250 mmol/L cCl - 30-900 mmol/L cCa 2+ 0.1-20 mmol/L Zero point pO2pO2 < 6 mmHg (0.8 kPa) cGlu< 10000 pA cLac< 10000 pA

17. 17 Sensitivity and Status - during measurements The sample pH is calculated as follows pH sample = pH in sample E sample = measured potential in sample E cal1 = measured potential in Cal 1 pH cal1 = pH in Cal 1 –61.5= sensitivity of theoretical calibration line

18. 18 Drift The drift of an electrode is a description of how much a calibration differs from the last calibration Drift is a check of whether the adjustment performed during calibration is acceptable This is defined by the Drift Tolerances A drift value of –0.001 pH units indicates that during calibration the analyzer has been adjusted to measure 1 mpH unit higher

19. 19 Calculation of drift Drift 1 reflects the change in position between two calibrations (given at both 1-point and 2-point calibrations) Drift 2 reflects the change in slope between two 2-point calibrations (given only at 2-point calibrations) 6.800 7.400 mV pH Slope of first 2P-cal. First 2P-cal. Second 2P-cal. Drift 1 Drift 2

20. 20 Drift tolerances Drift tolerances express the extent to which drift values for an electrode can fluctuate Drift values lying outside the tolerance limits indicate that the electrode is unstable and thus incapable of providing reliable calibrations Drift tolerances can be user- defined in the Setup programs, but should be done so with care Access from Calibration Setup

21. 21 Sensitivity of pO 2 electrode Obtained from a 2-point calibration using Gas 1 and 2 0.0 60 pA kPa 2P-cal. 18.6 2800  I 2800 – 60  pO 2 18.6 – 0.0 Sensitivity = Example: Sensitivity = = 147 pA/kPa

22. 22 pO 2 zero point Calculated from the sensitivity and the current measured at pO 2 = 0 0.0 18.6 60 2800 kPa First 2P- cal. Second 2P-cal. 85 0.58 I 0 85 I 0 85 Sensitivity, previous cal. 147 Sensitivity, previous cal. 147 Zero point = Example: Zero point = = 0.58 kPa pA

23. 23 Metabolite sensitivity Base line formed by extrapolation of consecutive zero-point currents measured on rinse solution Time I cal I0I0 I cal – I 0 5500 – 400 C Met 10.0 Sensitivity = Example, sensitivity, Glu = = 510 pA/mmol/L pA 5500 400

24. 24 Calibration of optical system Zero point measured during each calibration Cuvette path length, L, measured during tHb calibration A =  × c × L = absorbance = extinction coefficient = concentration of optically active substance in tHb calibration solution = cuvette path length

25. 25 tHb calibration It is recommended that a tHb calibration is performed manually every three months

26. 26 Summary pH, pCO 2, K, Na, Ca, Cl SensitivityStatusDrift pO 2 Sensitivity Zero point Drift Glu, Lac Base line SensitivityDriftOXI Zero point Cuvette path length

27. 27 Calibration status The Analyzer Status – Calibration screen displays the results of the most recently performed calibration of each type available

28. 28 Calibration Log Results from previous calibrations are viewed via the Calibration Log

29. 29 2-Point calibration result

30. 30 1-Point calibration result

31. 31 Calibration printout

32. 32 Radiometer Medical ApS, Åkandevej 21, DK-2700 Brønshøj, Tel: +45 38 27 38 27, www.radiometer.com Radiometer Training Center, December 2004