Biochemistry Lesson V3

2008 HORIBA ABX, All rights reserved.

BIOCHEMISTRY
HORIBA ABX
Training Center
2008

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

CLINICAL

BIOCHEMISTRY

Introduction
Substrate :

Enzyme :
Base of Biochemistry
Biochemistry principle
Introduction
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
FOOD
Waste
Liquid Puncture

Organism

Blood

Urine

Biological liquids
BLOOD : Structure

Liquid element 60%
Cell part 40% (Ht)

Biological liquids
Blood : liquid element

With or without anticoagulant

Serum

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

Urine
qualitative

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

CSF CerebroSpinal Fluid
Glucose status

Others punctures :
Knee puncture (synovial)
Uric acid

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

Colorimetry (measurement of absorbance)

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

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
Potentiometry
Techniques
Techniques
Potentiometry
Colorimetry (with spectrophotometer)
Cuvette with reactional mix

Io
(initial intensity)
It
(intensity after cuvette)
L
Detector
Techniques
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
Turbidimetry (immunoturbidimetry)
Specific protein
recognised Proteins
Specific
antibody
Techniques
Antibody coated to
Latex beads
Antibody/Antigene complex
(network)
Turbidimetry Latex (immunoturbidimetry)
Techniques
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
Substrates
Enzymes
Specific Proteins
Ions
Medicine Test
Turbidimetry Colorimetry Potentiometry

Techniques
Sample
(Substrate, enzyme,)
Technical Result (OD, )
Calculation mode
Technique
Calculation mode
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
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
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
Final Result
(concentration, enzymatic activity)
Calibration
Sample
Calculation Mode
Technique
Calibration
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
Different types of calibration :
Linear
Non linear
Enzyme Case (for Mira)
Calibration
OD
Concentration
Linear
F = Conc(known)/ OD Measured
Conc(known)
OD
Measured
Calibration
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
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
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
Final Result
(concentration, enzymatic activity )
Calibration
Sample
Calculation Mode
Technique
Control
(checking : reagent, machine, )
Control
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
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
Bone
Calcium, Phosphorus, ALP

Cardiac
CK, ASAT, LDH, Myoglobin

Renal
Urea, Creatinine, Uric Acid

Clinical biochemistry panel
Thank you

Biochemistry Lesson V3

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2008 HORIBA ABX, All rights reserved.

2008 HORIBA ABX, All rights reserved.

2008 HORIBA ABX, All rights reserved.

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