Principios de Medición

1
EL ABL 1

2
EL NORMOGRAMA DE SIGGAARD
ANDERSEN.(1963)

3
Radiometer ABL 800

4
Parameters
• Contiene 10 lugares para los electrodos en dos torretas
• Un lugar lo ocupa el electrodo de referencia
• Los otros nueve son los siguientes:

pH, pCO2, pO2, cK+, cNa+, cCa2+, cCl– , cGlucose, cLactate

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Electrode modules

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General construction
• The term ‘electrode’ refers to whole sensor unit

Preamplifier

Electrical contact

Color-coded ring
Electrode jacket (removable)

Electrolyte solution

Membrane
• Cordless – limiting electrical noise
• Amplifiers positioned in each module to amplify the electrical
signal
7
Metodos de medidas.
• El principio de la potenciometria es usado para :

pH, pCO2, cK+, cNa+, cCa2+, cCl–

• El principio de la amperometria es usado para:

pO2, cGlucose, cLactate

8
The potentiometric measuring principle
• Electrodes measure a change in voltage due to a change in ion
concentration across a membrane

pH, pCO2, cK+, cNa+, cCa2+, cCl–

V
Voltmeter
Reference electrode Electrode

Electrolyte Electrolyte Electrode

Electrode Sample solution
solution

Liquid junction Membrane

9
Measuring a potential
• Each link in the circuit exhibits a potential
• The only unknown potential is the one between the membrane
and the sample, ESample
• The potential of the whole circuit is measured, Evoltmeter, and the
unknown potential can be calculated:

E Sample
=Evoltmeter
- (E + E
Ref pH
)

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Reference electrode
• Provides a stable, fixed potential against which other potentials
can be measured
• The potential at the reference electrode is not altered by sample
composition
• A stable, fixed potential is obtained
by maintaining constant conditions
• The reference electrode is used in the
measurement of pH and the
electrolyte parameters

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The amperometric measuring principle
• Electrodes measure the current produced during an
electrochemical reaction at an electrode

pO2, cGlucose, cLactate

Applied voltage Ammeter

- measures current

Cathode Anode
- reduction - oxidation

Electrolyte
solution
- buffered

Sample
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pO2 and metabolite electrodes

• El electrodo de pO2 u los de metabolitos estan diseñados para

medir una corriente electrica producida en una reacción
electroquimica
An electrochemical reaction:
- a reaction where electrons are transferred

A+ + e-  A

• La corriente producida entonces, es proporcional a la

concentracion de los substratos ( pO2/Glu/Lac)

13
The metabolite membrane design

• Multilayered membrane
- outer layer permeable to
glucose/lactate
- middle enzyme layer (production of
H2 O 2 )
- inner layer permeable to H2O2

Inner layer
Middle layer
Outer layer

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Metabolites – measuring method

• Glucose/lactate molecules are transported across the outer

membrane to be converted by the enzyme to form H2O2

• The H2O2 is then oxidized to oxygen and electrons, a current,

which is measured by the ammeter

Glucose + O 2  Gluconic acid +H 2O 2

Lactate + O 2  Pyruvate +H 2O 2

H 2 O 2  2H+ + O 2 + 2e-

• The amount of current produced is proportional to the amount of

H2O2, which in turn is directly related to the amount of glucose
or lactate in the sample
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Metabolite membrane - outer layer
• Has pores of well-defined density and diameter to limit the
amount of glucose/lactate entering the enzyme layer
• Outer side is treated to prevent protein build-up that could block
the pores

Glucose/lactate

Inner layer Outer layer

Middle layer
Outer layer
Red blood cells

Blood sample

• The sensor is not affected by hematocrit due to the outer low-

porous membrane
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Metabolite membrane – middle enzyme layer

• The enzyme glucose or lactate oxidase is immobilized between

the outer and inner layer
• The enzymes only catalyze the following reactions:

Inner layer Glucose + O 2  Gluconic acid +H 2O 2

Middle layer
Outer layer Lactate + O 2  Pyruvate +H 2O 2

• O2 is supplied from the outer membrane to make the process

independent of the O2 content in the sample

Long lifetime of membrane

No dependency on sample’s oxygen content
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Metabolite membrane – inner layer
• The H2O2 from the enzyme reaction is transported across the inner
membrane to the Pt anode for oxidation

• The inner membrane is an interference-limiting membrane. In less than

30 seconds the H2O2 has diffused through the membrane and reached
the Pt anode.

Inner
Inner layer layer
Middle layer
Outer layer
Enzyme layer

E.g. Paracetamol-4-acetamidophenol

• Electrochemical substances that can interfere are delayed, e.g.

Paracetamol-4-acetamidophenol, and will not pass through the
membrane during the 30-second period

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Summary
• The following parameters are measured:
–
- pH, pCO2, pO2, cK+, cNa+, cCa2+, cCl , cGlucose, cLactate

• Two measuring principles

- ion-selective electrodes for pH, pCO2 and electrolytes
- measurement of current produced from reactions of O2, Glu and Lac

• Multilayer interference-limiting membranes for glucose and

lactate electrodes
- Long lifetime of membrane
- No dependency on sample’s oxygen content
- No interference from commonly known substances

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Que es una calibración?

• Un liquido o un gas de valores de cc conocidos son llevados

a la camara de medición.
• El electrodo mide una señal electrica correspondiente a este
valor
• El proceso se repite con un segundo gas o liquido tambien
de cc conocidas
• El analizador establece entonces una linea de calibración
para cada parametro Las mediciones de las muestras son
entonces interpoladas en esta linea de calibracion trazada.

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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
pCO2 and pO2 electrodes
• The tHb Calibration Solution is
used to calibrate the optical
system

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Why calibrate?
• Los electrodos son elementos
activos, y deben ser calibrados
en forma regular
• Las señales que llegan desde
los electrodos cambian debido a
:
- Deposito de proteínas
- Desgaste de las membranas
- Envejecimiento de los electrodos

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Curva de calibracion
• La curva de calibracion representa la relacion entre la señal
electrica medida por el electrodo y la concentracion del substrato
especifico

mV

–69

–106 6.800 7.400

pH
6.800 7.400

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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
mV

–69

–106

pH
6.800 7.400

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Obtaining a calibration line
• La relacion entre el potencial medido y el ph es lineal en el rango
de calibración y de medicion del electrodo
• La linea de calibracion me permite entonces, transformar en pH
un potencial medido.

mV

–69

–106

pH
6.800 7.400

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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

mV

–69

–106 2P cal.

pH
6.800 7.400

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Monitoring the calibration line
• La curva de calibracion es actualizada en cada ciclo de
calibración, y se monitorea respecto de los siguintes párámetros:

Sensitivity Describe la pendiente y la

Status/zero point posicion de la curva de
calibración.

Describe la variacion
en la curva de calibracion
Drift
entre dos consecutivas
calibraciones

Each electrode has its own status and sensitivity limits

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Sensitivity (slope)
• La sensibilidad es una evaluacion en % de la pendiente de la
calibracion actual respecto de la curva de calibracion teórica del
electrodo ideal.
- La curva teórica de calbracion deriva de la ecuación de Nernst.

mV

–69

Actual

–106 Theoretical

pH
6.800 7.400

• The theoretical pH calibration line has a slope = –61.5 mV/pH

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Sensitivity, an example
• During a 2-point calibration the following is measured:

- 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
mV

–65
–69

E

–101
2P-cal.
–106
pH
Theo.
pH
6.800 7.400

E –101.0 – (–65.0)
Sensitivity = pH × (–61.5) × 100 % Example: Sensitivity = (7.4–6.8)×(–61.5) × 100 % = 97.6 %

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ABL800 FLEX sensitivity limits

• For a calibration to be accepted, the sensitivity must fall within

the following limits:

Parameter Sensitivity limits

pH
pCO2
92-103 %
84-100 % E

pO2 5-40 pA/mmHg 2P-cal.
pH
(37.5-300 pA/kPa)
Theo.
cK+ 92-105 %
cNa+ 90-105 %
cCl-
cCa2+
85-105 %
90-105 % E
2P-cal.
%
cGlu 100-1800 pA/mM pH
cLac 150-2000 pA/mM Theo.

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Status
• El estado describe, sobre una cal de un punto, la
desviación de un punto determinado de esa calibracion
respecto del valor en la calibracion teórica.
- the status is evaluated using only one calibration point; i.e. we
assume that the sensitivity (slope) is unchanged
mV

–69

1P-cal.
–100 E
–106

Theo.
pH
6.800 7.300 7.400
E –100 – (–106)
Status = 7.40 + Example: Status = 7.40 + = 7.30
–61.5 –61.5
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ABL800 FLEX status limits
• For a calibration to be accepted, the status and zero point
must fall within the following limits

Parameter Status limits

pH 6.7-8.1
pCO2
cK+
6.2-260 mmHg
0.5-12 mmol/L cal.

cNa+ 10-250 mmol/L
Theo.
cCl- 30-900 mmol/L
cCa2+ 0.1-20 mmol/L
Zero point cal.
pO2 < 6 mmHg
(0.8 kPa)
%
cGlu < 10000 pA Theo.

cLac < 10000 pA

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 Deriva (Drift)
• La “deriva” de un electrodo es una medida de cuanto esa
calibracion difiere de la anterior
• La deriva tambien me permite chequear si el ajuste realizado en
la ultima calibracion es aceptable.
• Esto es definido por el manual de acuerdo a tolerancias de deriva

A drift value of –0.001 pH units indicates that

during calibration the analyzer has been adjusted to
measure
1 mpH unit higher

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Sensitivity of pO2 electrode
• Obtained from a 2-point calibration using Gas 1 and 2

pA

2800 2P-cal.

60
kPa
0.0 18.6

Sensitivity =I Example: Sensitivity =2800 – 60 = 147

pA/kPa pO2 18.6 – 0.0
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pO2 zero point

• Calculated from the sensitivity and the current measured at pO2 = 0

pA

Second 2P-cal.
2800
First 2P- cal.

85
60
kPa
0.0 0.58 18.6

Zero point = I0 Example: Zero point = 85 = 0.58 kPa

Sensitivity, previous cal. 147
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Metabolite sensitivity
• Base line formed by extrapolation of consecutive zero-point
currents measured on rinse solution
pA

5500

Ical

400
I0
Time

Ical – I0 5500 – 400

Sensitivity = Example, sensitivity, Glu = = 510 pA/mmol/L
CMet 10.0

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 ABL800 FLEX
Quality control

RTC, December 2004

Radiometer Medical ApS, Åkandevej 21, DK-2700 Brønshøj, Tel: +45 38 27 38 27, www.radiometer.com
37
Why use quality control

• Tool for early error detection

• Alerts when maintenance is
needed
• Validation of analyzer
performance by evaluating
- Accuracy: all results must fall
within the defined control
ranges
- Precision: no trend or shifts in
the plotted QC results
• This can only be done by
measuring on control
materials with
predetermined values
- not by duplicate testing
- not by calibration

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AutoCheck - the no-worry solution

• The purpose of quality control is

- to check the performance of the
analyzer
- not to check how good the operator is
at introducing QC solution
• AutoCheck makes QC
measurements more uniform
and eliminates the risk of user
bias on QC results
• Reliable QC results are a
condition for fulfilling the
purpose of quality control
• More value for the time/money
spent on quality control
• A good investment for several
reasons
- e.g. no extra ampoules for repeated
QC measurements due to operator
errors
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What can go wrong during a manual QC measurement?

• La ampolla no se acondiciona correctamente antes de su uso:

• Ampolla no es agitada vigorosamente durante al menos 15
segundos
• Ampoulla se calienta mientras es preparada
• Se retrasa la medicion luego de haber roto la ampolla
• No se usa un adaptador para la medicion de la ampolla
• Se desconoce la temperatura de la misma al momento de la
medicion.

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FORTALEZAS DEL QC DE RADIOMETER

• Mediciones a traves de muestras unicas.

• Reproduce la medición tal cual una muestra:
• Misma puerta de entrada
• Mismo volumen sampleado
• Mismo tiempo de permanencia en la camara de medicion

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 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
42
Location

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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

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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

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Measuring principle
• 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

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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

Photodiode array Concave grating

Slit

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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
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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

Absorbance Absorbance
A
B

Wavelengt Wavelengt
h h
Absorbance A+B

Wavelengt 49
Lambert-Beer’s law
• Measured absorbance of a single substance is directly
proportional to its concentration

l l
A y = y  c y  l
A ly = absorbance of substance y at wavelength l
 yl = extinction coefficient of substance y at
wavelength l
cy
= concentration of substance y in sample
l
= length of light path
l
Note that sometimes  y  l is given as a single constant

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Absorption of blood
• The absorption spectrum of a blood sample is a summation of all
the different hemoglobin spectra
Absorbance

HHb
1.5 O2Hb
COHb
MetHb
SHb
Bilirubi
n

500 550 600 650 700 nm

51
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

52
Calculation
• 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

cO Hb
Example: F O2Hb = 2
ctHb

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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

A = absorbance
 = extinction coefficient
c = concentration of optically active
substance in tHb
L Calibration solution
= length of light path

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 CAPTURA DE 02
pH pCO2 pO2 EN LOS
ctHb PULMONES
So2
FO2Hb
FCOHb TRANSPORTE DE
FMetHb 02 EN LA SANGRE
ctO2
p50
LIBERACIÓN DE
02 A LOS
TEJIDOS
DESCRIBE LA CAPACIDAD DE LA SANGRE ARTERIAL PARA
PROPORCIONAR 02 A LOS TEJIDOS
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 Oxigenación

En el paciente críticamente enfermo, la adecuada

oxigenación para prevenir hipoxia tisular es uno de los
parametros esenciales para ayudar a su recuperacion

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 ¿Qué hay que tomar en cuenta en el
proceso de oxigenación?

La Captación de oxígeno

-La transferencia del oxígeno atmosférico a los
pulmones.

El Transporte de Oxígeno

-Los mecanismos de transporte del oxigeno
en la sangre a los tejidos

La Cesión de Oxígeno

-La difusión del oxígeno de la sangre hacia los
tejidos

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 paO2,
pA O2 = FiO2 x(pBar – 47) – pCO2/0,8
a/A : ( V Ref0,85 – 0,95)
SO2: HbO2 / (HbO2 + HbH)

CAPTURA DE
02 EN LOS
PULMONES

58
CaO2 = p.OXI x(Hb)x HbO2,x0,003xpO2
CvO2= idem en sangre venosa mixta
Dif (a-v)= CaO2 – CvO2

TRANSPORTE DE
02 EN LA
SANGRE

59
P50
pvO2:
Px: tensión de O2 extraible
ErO2: fracción extraída de O2 =
VO2/ DO2

CESIÓN DE 02
A LOS TEJIDOS

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 sO2

0.9

0.8
2.3 - DPG 0.7 2.3 - DPG
 
0.6
Temp.  Temp. 
pCO2  0.5
pCO2 
pH  0.4
pH 
FHbF  0.3
FSHb 
p50
FCOHb 0.2
FMetHb
0.1 pO2
20 40 60 80 mmHg
0
LA CAPTURA Y LIBERACIÓN
0 2 4 6 8DEL
10 02
12 POR LA Hb ES
kPa

EXPRESADA POR LA RELACIÓN ENTRE p02 Y s02

61