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Photometric Calibration

Roche User Group 2018Presented by Caroline Roche

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Photometric Calibration

• Photometric calibration curves

• Photometric calibration quality checks

• Photometric calibration methods

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Photometric calibration curves

Linear calibration curve

• Assay absorbances at differentconcentrations form a linear plot

• Linear 2-point

• Linear multipoint (min 3 max 6 standards)

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Photometric calibration curves

Non-linear calibration curve

• Plot of assay absorbances at differentconcentrations form a nonlinear curve

• Multipoint calibration

• Min 3 max 6 standards

• RCM, RCM2T1, RCM2T2, Spline, Line Graph

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Photometric calibration curve parameters

• Calibration curve is mathematically defined by its curve parameters

• In the user interface these are defined as S1 Abs, K, A, B, C

• Linear calibration curve – defined by two parameters S1 Abs and K

• Non linear calibration curve e.g. RCM defined by four parameters

• When an assay is recalibrated software plots the calibration curve using the newly measured values and recalculates these parameters

• Curve parameters displayed in Calibration Status > Calibration Result > Working Information

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Photometric calibration curve parameters

Linear two-point:

S1 Abs = Mean ABS of STD1 Blank

K = K Factor

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K factor (calibration factor)

• K factor is a calibration curve parameter calculated for linear curves.

• Ratio of the difference between the concentration of standards and the difference between the absorbance of the standards.

• For example for two point linear calibration for an end point assay:

K = (Concentration STD2 – Concentration STD1 (blank)) / (Abs STD2 – Abs STD1(blank))

• Used to calculate results from test absorbances

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K factor (calibration factor)

Calibration Working Information Screen – K factor

Always displayed as whole number

Automatically rounded and multiplied by the correct factor of 10 according to the number of decimalplaces set for STD 1 (blank)

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Photometric calibration curve parameters• Example – 2 point linear calibration

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Photometric calibration curve parameters

RCM non-Linear Multipoint

S1 Abs: curve parameter a

K: curve parameter b

A: curve parameter c

B: curve parameter d

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Photometric calibration curve parameters• Example – RCM non linear multipoint calibration

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Photometric calibration quality checks

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Photometric calibration quality checksSD Limit

Non-linear and multipoint linear calibration curves

Absorbance is calculated from givenconcentration using calibration curve for eachstandard and is compared to measuredabsorbance

SD.E alarm generated if exceeds SD limit

Affected standard is flagged on calibration monitor

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Photometric calibration quality checks

SD Limit

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Photometric calibration quality checks

Duplicate Limit

Precision check on duplicate measurements

Calculates % error and absolute absorbance errorbetween duplicate calibrator measurements

Evaluates absolute error first, if this is out of range evaluates % error

Dup.E alarm generated If both out of range

Affected standard is flagged on calibration monitor

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Photometric calibration quality checks

Sensitivity Limit

Ratio of the absorbance difference to the concentration difference

(ABS Span STD – ABS Blank STD) / (Conc SpanSTD – Conc Blank STD)

Upper and lower limit defined in Application/Calib screen

Sens.E alarm generated

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Photometric calibration quality checks

S1 Absorbance Limit

Check on blank calibrator absorbance

S1 A.E Alarm generated if falls outside limits

Range of -32000 to 32000 indicates that thischeck is omitted

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Photometric calibration quality checks

Std. Check

Std.E alarm issued if any of the listed data alarms occur in a calibration (cobas 6000)

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Photometric calibration quality checks

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Calibration methods

2 Point

Full

Blank

Span

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Calibration methods

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Span CalibrationUpdate Type – Difference

Absorbance difference for the measured calibrator is added to each calibrator’s value. Calibration curve is moved up or down maintaining slope.

Update Type – Rate

Ratio of new absorbance value to previous yields an adjustment factor. Each calibrator absorbance is multiplied by this factor.

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