Biochemistry Fundamentals

--IVD International Clinical Application Department

 Course Overview

• Chemistry Function

• Chemistry Classification

• Chemistry Working Principle

• Chemistry Calibration & Quality Control

• Q&A

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 Course Overview

• Chemistry Function

• Chemistry Classification

• Chemistry Working Principle

• Chemistry Calibration & Quality Control

• Q&A

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 Basic Knowledge - Sample Type

Plasma Serum

Anticoagulant Yes No

Fibrinogen Yes No

Plasma and Serum

Urine
CSF

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 Basic Knowledge - Major Menu
ALT, AST,GGT, UREA, UA, CK, CK-MB, a- Glu, HbA1C,
ALP, ALB, TP, CO2, Cys-C, HBDH, LDH, β-HB, FUN
BIL-T, BIL-D, β2-MG, RBP, HS-CRP, MYO,
PA, TBA, CHE, MALB, CREA D-Dimer
AFU,5’-NT,ADA TPUC

Liver Kidney Myocardium Diabetes

TC 、 TG 、 IgG 、 IgA 、 CRP 、 ASO α-AMY 、 LPS

HDL-C 、 LDL- IgM 、 IgE 、
C 、 ApoA1 、 C3 RF
ApoB 、 Lp(a) C4

Lipids Immunology Rheumatism Pancreas

CO2 、 FER, ADA ACE

Ca 、 P 、 Mg UIBC, TRF,
、 Fe 、 G6PD
TDM/DAT

Ions Anemia Thrombus Pulmonary Infarction

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 Course Overview

• Chemistry Function

• Chemistry Classification

• Chemistry Working Principle

• Chemistry Calibration & Quality Control

• Q&A

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 Basic Knowledge - Classify

Mode Auto

 Wet Mode  Semi-Auto

 Dry film  Automatic
 TLA

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 Basic Knowledge - Classify by Mode

Flow cell mode

Peristaltic pump

Wet mode

Discrete mode

Dry mode

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 Basic Knowledge - Classify by Speed

<300T/H 300T/H-1000T/H 1000T/H -2000T/H

Up to 8800 T/H

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 Basic Knowledge - Classify by Automation

Semi-Auto Automatic Total Lab Automation

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 Course Overview

• Chemistry Function

• Chemistry Classification

• Chemistry Working Principle

• Chemistry Calibration & Quality Control

• Q&A

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 Basic Knowledge – Elements of Biochemistry System

Analyzer & Controller

Accessories kit Reagent ， CAL ， Q Barcode reader and
(LIS is optional & locally)
C, Detergent (e.g. ISE modular
CD80) (Optional)

Water system & Water supply unit

UPS >=4KVA Spare parts, maintenance kit
(Locally) (very important)
(Locally)

Optional: main function won’t be influenced without the module

Locally: purchase from local market, Mindray won’t supply

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 Test Process

Add

R1 Report

(The first step)

Add Mixing Reaction Data
Mixing Incubation Reaction
R2 Incubation monitoring processing
Add
Cleaning
sample

(The second step)

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 Add Sample Add R2

Incubation time
Incubation time

Add R1

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 Basic Knowledge - Measuring Principle
How to measure the concentration of the solutions?

Measuring principle

• Colorimetric Method

• Turbidimetry and Nephelometry

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 Basic Knowledge - Colorimetric Method
Beer-Lambert Law

Absorbance: in spectroscopy, the absorbance A is

defined as A=-log(/)

: the intensity of the light before it enters the sample or

incident light intensity.

: the intensity of light that has passed through a sample

(transmitted light intensity).

Beer-Lambert Law: the absorbance becomes linear with the concentration A=εcl, where l is the
distance the light travels through the material and ε is the absorption coefficient of this material.

Precondition: weak solution and monochromatic light.

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 Basic Knowledge - Immunoturbidimetric Assay Method
Antigen and Antibody interaction Turbidimetry and Nephelometry

C3, C4, IgA, IgG, IgM, IgE

Apo-A1, Apo-B

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 Basic Knowledge - Working Principle

1. Endpoint

2. Kinetic

3. Fixed-time

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 Basic Knowledge - Working Principle
End point

• The reaction reaches equilibrium after certain period.

• The absorbance change is proportional to the amount of the product present.
• E.g. TP, UA, Glu, HbA1c, TC …

A equilibrium

t1 t2 t3 t

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 Basic Knowledge - Working Principle
Kinetic
• The reaction velocity remains constan during the process.
• The absorbance of the reactingt liquid changes evenly, which is proportional to the activity of the
subject (usually the enzymes).
• E.g. ALT, ALP, LDH, CK, ACE …

A Constant
velocity

t1 t2 t3 tn
t
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 Basic Knowledge - Working Principle
Fixed-Time
• It takes considerable time for the fixed-time reaction to reach the equilibrium.
• In specific period of the reaction, the reaction velocity (v), is proportional to the substrate
concentration [S].
• E.g. ASO, CO2

Specific
period

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 Course Overview

• Chemistry Function

• Chemistry Classification

• Chemistry Working Principle

• Chemistry Calibration & Quality Control

• Q&A

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 Basic Knowledge - Calibration & Quality Control

1. Calibration

2. Quality control

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 Basic Knowledge - Calibration
Purpose
• Get relations of Response and analyze Concentration
• Standardization

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 Basic Knowledge - Calibration
Linear calibration: used for those analytes (e.g., TP, ALB, and TC) whose assay absorbance/Absorbance
change at different concentrations form a linear plot.
• Single-point linear;
• Two-point linear; R R=aC+b
• Multi-point linear.
R=aC

Non-linear calibration: used for those sample constituents (e.g. specific proteins,
and Immunology tests IgM, IgE) whose assay absorbance at different concentrations C
do not form a linear plot.

• Logit-Log 4P/5P;
• Exponential/ Polynomial 5P; R=Xa
R
• Parabola;
• Spline.

C
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 Basic Knowledge - Calibration
Linear calibration:
Calculation formula: C= K×(R-R0). R0 is the reagent blank response.

One-point calibration

Two-point calibration

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 Basic Knowledge - Calibration
Non-Linear calibration:
• Logit-Log 4P: the calibration math model requires at least four
calibrators. The four factors can be calculated with the L-M method.
• Logit–Log 5P: this math model has the same application with the Logit-Log 4P
except for a higher fitting.
• Exponential 5P: this calibration type is applied to the chemistries which
have a calibration curve with the response directly proportional to the
concentration.
• Polynomial 5P: the calibration math model requires at least five
calibrators.
• Parabola: the calibration math model requires at least three calibrators. The
three factors can be calculated with the least square method.
• Spline: the calibration math model requires 2-9 calibrators. Due to the
subsection fitting, this math model has be best fit curves than other math
models.

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 Basic Knowledge - Calibration
Pre A/D
Amplification Conversion
Board Board
Light Electrical Digital
Analog Signal / AD
Absorption Signal value

A = log10 P0 / P
Endpoint

Response Calculated
Kenetic
R=ΔA Absorbance

Fixed-time

Linear
Calibration Concentration
Curve C = K × (R-R0)
Non-linear

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 Basic Knowledge - Quality Control

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 Basic Knowledge - Quality Control
Westgard rules: Multirule QC uses a combination of decision criteria, or control rules, to decide
whether an analytical run is in-control or out-of-control.

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 Basic Knowledge - Quality Control
• Internal Quality Control (IQC)

• External Quality Assessment(EQA)

– Randox RIQAS
– Biorad EQAS
– CAP
…

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 Basic Knowledge - Clinical Testing System

The fixed combination of

Analyzer, Reagent and
Calibrator is the premise
to realize the traceability.

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 Matching AAA System with Full Traceability

Instrument, Reagent,
Level Remark
Calibrator

Analyzer, reagents and calibrators

Matching system with
AAA are from the same brand,
system integration,
System registered as systematic solution,
ensure the accuracy
and the system has traceability
Analyzer, reagents and calibrators
Open system without
ABB or are from different brands, not
system integration, can’t
ABC registered as systematic solution,
ensure accuracy and
System the performance has no
comparability
verification
Remark: A, B, C represent for different company’s instrument, reagent,
calibrator

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 Course Overview

• Chemistry Function

• Chemistry Classification

• Chemistry Working Principle

• Chemistry Calibration & Quality Control

• Q&A

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 Q1 Why the detection wavelength is same for AST & ALT?

• Because they using same indicator

system: NADH/NADPH. Which has
absorbance peak at 340nm.

• NAD+→NADH (NADP+ → NADPH)

ALT

AST

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 Q2 How many color indicators we have?

 NAD+→NADH (NADP+ → NADPH)

 Phenolic Salts → p-Nitrophenol (405nm)
Example: ALP (IFCC reference method )

 H2O2 & 4-Aminoantipyrine (546nm)

Example: UA (Uricase –POD method)

 Union of Antigen & Antibody → Immune complex

Example: C4 (Turbidimetric assay method)

 Other coloration reaction: Ca, Mg …

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 Q3 What’s the advantage of dual wavelength?
1. Benefit of Dual Wavelength — Eliminate non-reaction interferences

Restrain noise

Amend shift
Reduce fluctuation

Prevent cuvette diff.

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 Q3 What’s the advantage of dual wavelength?
2. Benefit of Dual Wavelength - Eliminate endogenous interferences

 Suppose component X is determinand, while X Y

component Y is the main distraction. A
 λ1 and λ2 are the dual wavelengths we used.
 We use ΔA to calculate absorbance value
A  A1  A 2  ( Ax1  Ay1 ) （Ax2  Ay2）
According to the absorbance curve, we get:
y y
∵ A1  A 2
1 2 
∴ A  Ax1  Ax 2
X: determinand
Conclusion: Y won’t interfere the measurement of X Y: distraction

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 Q4 Why there are constant speed and inconstant speed for
different analyzer?

• There are 4 main reasons limit throughput:

– Optical system, normally grating system has higher efficiency than filter system.
– Wash system, system without wash station asks break for cuvette replacement.
– Incubation position, small reaction disk will be saturated easily.
– Continuously loading, those devices with limited probe number won’t have
enough time to finish S/R1/R2 injection in one period.

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 Q5 What is the benefit if we start with R1 not S?

If starts with S (sample) …

If start with R (reagent):

• Avoid inaccurate sample injection due to
liquid drop on sample probe needle end
• Probe will check liquid level of R1 before
injection, in order to verify R1 dispense

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 Key Points Summary

• From light absorbance to sample concentration based on Beer-

Lambert Law

• Calibrator is used to establish R-C (Response-Concentration) chart

• Quality control is used to monitor system performance

• AAA system is full traceability.

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Thank you!