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2008 HORIBA ABX, All rights reserved.

2008 HORIBA ABX, All rights reserved.


2008 HORIBA ABX, All rights reserved.
BIOCHEMISTRY
2008 HORIBA ABX, All rights reserved.
HORIBA ABX
Training Center
2008

2008 HORIBA ABX, All rights reserved.
Biochemistry = Chemistry of life
Introduction
Biochemistry is a scientific discipline which
explore, in human being, chemical reactions
allowing the maintenance of the living status.
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Water
Glucids
Lipids
Proteins
Mineral salt
Others
Substrates
Specific Proteins
Enzymes
Ions
Medecine
Drugs
TDM
DAT/DAU



CLINICAL


BIOCHEMISTRY



Introduction
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Substrate :

Enzyme :
Base of Biochemistry
Biochemistry principle
Introduction
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Substrate : Molecule or substance which undergo or take action in chemical
reaction. After reaction this substrate give a product.
Enzyme : Protein substance,which catalyse chemical reaction. The action
of enzyme is substrate specific and action specific (... ase)
.



Specific Protein : Protein with immunogenic properties (characteristics).
They can be selectively isolated by Immunoturbidimetry
(antibody)

DAT / TDM : Drug of Abuse Testing. &Therapeutic Drug Monitoring
(for medicine or drug tests).


Ions : Electrolytes, mineral compound in biological liquid

Introduction
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FOOD
Waste
Liquid Puncture

Organism

Blood

Urine

Biological liquids
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BLOOD : Structure

Liquid element 60%
Cell part 40% (Ht)


Biological liquids
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Blood : liquid element

With or without anticoagulant


Serum


Plasma
Clot (Fibrin +
thrombin +
coagulation
factors)
Ht
Biological liquids
30 min
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Urine
qualitative

Urine in 24 h (1day)
quantitative & qualitative
additives
Biological liquids
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Liquid puncture

CSF CerebroSpinal Fluid
Glucose status

Others punctures :
Knee puncture (synovial)
Uric acid






Biological liquids
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Five type of tests
Substrates
Enzymes
Specific Proteins
Ions
Drug testing
Different
techniques
Techniques
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Potentiometry (measurement of voltage)

Colorimetry (measurement of absorbance)

Turbidimetry (measurement of level of opacity, cloudy)
Techniques
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Techniques
Potentiometry
+
Voltage
measurement
Electrode
Reference
Selective electrode
(for the ion measured)
Selective Membrane
Electrolyte
(known concentration)
+
Voltage
measurement
Electrode
Reference
Selective electrode
(for the ion measured)
Selective Membrane
Electrolyte
(known concentration)
+
+
+
+
+
+
+
+
+ +

++
Voltage
measurement
Electrode
Reference
Selective electrode
(for the ion measured)
Selective Membrane
Electrolyte
(known concentration)
++
++
++
++
++
++
++
++
++ ++

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Voltage measurement between a selective electrode and
a reference electrode.

The intensity is proportional to the selected ion (by the
selective electrode : Na, K, Cl,...).
Potentiometry
Techniques
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Potentiometry
Techniques
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Techniques
Potentiometry
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Colorimetry (with spectrophotometer)
Cuvette with reactional mix




Io
(initial intensity)
It
(intensity after cuvette)
L
Detector
Techniques
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T = It / I0 (Transmittance)
A = OD = log(1/T) = log(I0/It) (Absorbance)

Each molecule have a specific coefficient of molecular
absorption for one wavelength

Beer-Lambert law : OD = .C.L
L = length or path of light
C = compound concentration


Colorimetry (with spectrophotometer)
Techniques
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Turbidimetry (immunoturbidimetry)
Specific protein
recognised Proteins
Specific
antibody
Techniques
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Antibody coated to
Latex beads
Antibody/Antigene complex
(network)
Turbidimetry Latex (immunoturbidimetry)
Techniques
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photometry of cloudy solution (Rayleigh law) :

I0 incident = I absorb + I transmit + I diffuse

(proportional to the amount of specific protein
recognised)



Turbidimetry (immunoturbidimetry)
Techniques
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Substrates
Enzymes
Specific Proteins
Ions
Medicine Test
Turbidimetry Colorimetry Potentiometry







Techniques
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Sample
(Substrate, enzyme,)
Technical Result (OD, )
Calculation mode
Technique
Calculation mode
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Method with final point = endpoint
ex : Cs, TG, uric acid

OD
OD f
final
OD i
initial
Time
OD in end of reaction
Substrate
concentration

OD measured = OD
final
- OD
initial

Calculation mode
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OD
Time

OD

T
Kinetic with fixed time = kinetic
OD/min measured is proportional concentration (substrate) or enzyme
activity (enzyme)


ex : urea, creat., glu GDH
Calculation mode
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OD
Time

OD

T
Kinetic with research of linear zone = kinsearch
Measure of slope (OD/min) of linear zone (at leats 5 points in the same straight),
proportionality with concentration (substrate), or enzymatic activity (enzyme)
Calculation mode
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Final Result
(concentration, enzymatic activity)
Calibration
Sample
(Substrate, enzyme,)
Technical Result (OD, )
Calculation Mode
Technique
Calibration
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Aim : give a relation between the signal/result measured (Absorbance)
and the concentration or the activity measured

Calibration curve : OD = f(Conc)

Calibrator = standard serum or solution with known concentration
substance

Calibrator mono & multiparametric

Calibrator alone (for linear calibration)
Multi Calibrator (non linear Calibration, algorithm)


Calibration
Calibration
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Different types of calibration :
Linear
Non linear
Enzyme Case (for Mira)
Calibration
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OD
Concentration
Linear
F = Conc(known)/ OD Measured
Conc(known)
OD
Measured
Calibration
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Linear
1- Slope average mode (Slop Avg)
ODmeasured using the calibrator allow to calculate the calibration factor :
F = Conc(known)/OD Measured

After for each Sample, you have :
Conc = F X OD
2- Linear regression mode (lin reg)

Conc = F X OD + Ro (Ro = offset)
Calibration
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Non linear


The measurement is not proportional
with the concentration or the activity

Calibration curve is given by different
types (concentration) of calibrators

Ex. :
LIN INTER ; LOGIT/LOG 4 ; LOGIT/LOG 5 ;
EXPONENT 5
N L
conc
s
i
g
n
a
l

Calibration
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Enzymatic activity (Mira case) :

The activity of each enzyme is known

you dont need calibration, you know directly the
FACTOR
Remark : F = Vol Total/(Vol Sample * d * )
d = optical path ( 0,6 cm)
= coefficient of molecular absorption
This factor is known for each method of analysis ( SFBC, DGKC, IFCC)

Enz Activity = OD/Time x F
Calibration
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Final Result
(concentration, enzymatic activity )
Calibration
Sample
(Substrate, enzyme,)
Technical Result (OD, )
Calculation Mode
Technique
Control
(checking : reagent, machine, )
Control
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Aim :
ensure the validity of calibration curve and reagent
Used Material :
Serum control mono or multiparametric
1 or 2 level of control (Normal + Pathologic)
Realisation :
Control tittered as a Sample
Target values with a confidence range
Obtained results :
Measuring value = theoretical value : Cal OK. Run the patients
Measuring value theoretical value : Cal OUT
Pb reagent
Calibrator Pb
Bad control
Instrument failure
Control
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Hepatic panel
Total Bilirubin, Direct Bilirubin, Transaminase ASAT &
ALAT, ALP & GGT
Glucidic panel
Glucose, HbA1c, Fructosamine, Micro Albumin
Lipidic panel
Cholesterol, HDL Chol, LDL Chol, Phospholipid,
Trigly, Apo A1, Apo B.
Clinical biochemistry panel
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Bone
Calcium, Phosphorus, ALP

Cardiac
CK, ASAT, LDH, Myoglobin

Renal
Urea, Creatinine, Uric Acid

Clinical biochemistry panel
2008 HORIBA ABX, All rights reserved.
Thank you