Technical Data BC-5300 - Five Part Diff-NA

Optical System
International Service Department

Contents
1. 5DIFF Principle
2. Optical System Structure
3. Adjustment
4. Scatter gram
5. Troubleshooting
Blood Cells Neutrophil
Eosinophil
RBC
Monocyte
PLT
Basophil
Lymphocyte
Normal blood cell volume (size) range :
Version: 1.0, Update Date: 2008-5-29
RBC : 60-120 fl , WBC: 120-1000 fl , PLT : 2-30 fl
Morphological

Conclusion:

5Diff Principle
4-DIFF channel: Lymph, Mono, Neut, Eos:
Flow cytometry
Laser scatter
Morphological treatment: DIFF channel
4 Diff scatter gram
BASO/WBC channel:
Impedance method
Morphological treatment: WBC channel
WBC histogram

Flow Mode of Cell Suspension
Normal flow Sheath flow

Hydrodynamic Foucsing (Sheath Flow)
 Sheath fluid surrounds and forces blood cells suspended in dilution to
pass through the center of flow cell one by one.
• Blood cells pass through the facula of flow cell with high speed.
• Sample flow width 20-30um.
20~30 um
Multi-angle Laser Scatter
High Angle Scatter 8~20
reflects granularity and
5
complexity
3 4
Low Angle Scatter 1~5
reflects the cell volume
1 Sample
2 Laser light
3 Lens
1
Laser Scatter: Mie Scatter
Mie scatter:
 Cells are bigger than the laser wave length.

Mie Theory
 For different cells of different size
 Low angle can better reflect the information of size
Laser
LAS

Low Angle Scatter (LAS)
The low angle scatter signal (1~5) along the direction of incidence light
is called forward scatter.
It reflects the volume of cell.

Mie Theory
 For different cells of same size

 High angle can better reflect the information of structure
Laser
MAS

4-DIFF Channel-Side Scatter
Laser
FS
SS
The side angle (8~20) scatter signal along the direction of incidence
light is called side scatter.
It is related to cellular contents (granularity, nuclear content, and so

forth ). It reflects the internal complexity of cell.
Flow cytometry + Laser Scatter
 Flow cytometry + Laser Scatter

FAQ
 Is Flow cytometry + Laser Scatter enough
to have 5-part?
Morphological treatment

4-DIFF Channel- 4 diff differential
Blood
LEO(I) RBC
Lym Gran Mon
LEO(II)
Eos Neu Bas
Dilute ratio 1:135(348)

Laser scattering technology
DIFF Bath FS
RBC Lymphocyte Monocyte Basophil Eosinophil Neutrophil
LEO I Lyse
SS
LEO II Lyse
?
Leo II lyse penetrates the eosinophils membrane and binds the granules to increase complexity;
Flow cell
Semi-conductor laser
DIFF channel
Nucleus Basophile
Scatter Scatter
intensity intensity
Time Time

DIFF channel
SIZING STAIN
Eos
Neu
Bas
... . .. . ... . .. . ... . .. . Mon

. .. . .. . ..
. . Lym
.

4-DIFF Channel-Scatter gram
Neu
Mon
Eos
Lymph
Ghost
Horizontal axis: High angle scatter signal reflects the internal complexity of
cell.
Vertical axis: Low angle scatter signal reflects the volume of cell.

BASO/WBC channel-Impedance
Method Pulse graph
40fL
35fL
30fL
25fL
Diluent 20fL
+ LH
Aperture Oscillograph
Histogram

BASO/WBC channel-Baso differential
Blood
LH RBC
Baso Lym, Mon, Neu, Eos

BASO channel
SIZING
Eos
Neu
Bas
... . .. . .... .. Mon

. .. ..
. Lym

Differential lyse for Basophils
WBC/BASO Bath
RBC Lymphocyte Monocyte Basophil Eosinophil Neutrophil
LH Lyse
525nm
HGB
LH lyse shrinks the white blood cells to reduce the size except for basophils;
Baso/WBC Histogram

Optical System Structure

Optical System Structure
Note: MAS=SS LAS=FS

Optical System Components
Laser control board
SS PD Assembly
FS PD Assembly
FS Beam Stop
SS Beam Stop
Flow Cell
Assembly
Back Lens Assembly Splitter
Forward
Version: Optical
1.0, Update Assembly
Date: 2008-5-29 Backward Optical System
Forward Optical Assembly
■ Function: generate facula to irradiate the blood

cell.

Forward Optical Assembly

Laser Source
Semiconductor laser technology
small
Semiconductor – more stable, low q
uantity of heat
long life and low cost for
maintenance

Flow Cell Assembly
■ Function: generate stable sample flow; make
blood cell in the sample flow pass trough laser
irradiation area one by one.

Sheath fluid bath

Sheath fluid observation

Backward Optical System
■ Function: first collimate the scatter light, then use prisms
to separate the light into two directions, and use beam stop
in each direction to collect the light at certain angle.

Backward optical system optical path

Back lens assembly
• Block the direct light, and then collimate.

Splitter assembly
• Divide into two directions.

Beam Stop Assembly
• Block the scatter light at certain angle, and then focus
onto the PD.
SS Stop Assembly
FS Stop Assembly

PD Assembly
• Remove the stray light and convenient to focalize.

Adjust Focus Position
Let pulse of SS be biggest.
Oscilloscope settings ： AC Coupler , Voltage 100mv/div ， Time 500ns/div
2
2

Scatter gram and histogram

Normal Scatter grams and histogram

Normal scatter gram of standard particle
Standard particle target value
Param Before version After version
1.4 1.4
FS 26 ± 3 18±2
SS 117± 5 104±6
SS 0.1Max ≤18 ≤15

Width
FS 0.1Max ≤13 ≤8
Width
7um particle

Flow cell clog and dirty

Sample aspiration
Diluent Diluent
Air Air
4ul drain out
180ul diluent
6uL: WBC/HGB bath 40uL: WBC/HGB bath
20ul blood
40uL: DIFF bath

10uL: DIFF bath
Whole blood mode-20uL Pre-diluted mode

Sample distribution

Liquid Valve

Release valve

Release valve
Outlet
Spring
Movement
part Adjustment
screw
Membrane
Lock screw
Inlet
Optical
Trigge sensor
r

Pinch valve
We use only the NO port, but a

tubing must be placed in the
NC port to make sure the valve
work correctly.
NC
NO

DIFF Bath
LEO1 IN
LEO2 IN
To Flow cell

DIFF Bath
LEO1 IN
LEO1
LEO2
LEO2 IN

Flow Cell Bath

Application-WBC 4 DIFF



Syringes
10ml
2.5ml
100ul
250ul

Application of Syringes

Clean sample probe(inside)

WBC&RBC Baths

Pumps

Vacuum and pressure chamber
Note: The vacuum and pressure

chamber are the different part
number.

Application
P1:Bubble mixing
P2: Waste, Vacuum
P3: Waste

Volumetric tube

WBC(BASO)/HGB & RBC Bath

Application-WBC/BASO-Vacuum established

Application-WBC/BASO-Clean Counting bath

Application-WBC/BASO-Empty volumetric tube

Application-WBC/BASO-Counting

Application-WBC/BASO-Cleaning

Application-WBC/BASO-Cleaning

Application-RBC/PLT-Clean counting bath

Application-RBC/PLT-Empty volumetric tube

Application-RBC/PLT-Counting

Application-RBC/PLT-Cleaning

Application-RBC/PLT-Cleaning

Counting Principle
WBC channel
 Diff Channel: Laser + FCM
 Baso Channel: Electrical Impedance Method

Counting Principle
HGB channel
 Colorimetry

Counting Principle
RBC/PLT channel
 Electrical Impedance Method

Counting Sequence

WB Mode 0.15ml
Flow cell
LEOII
DIFF
36°C
LEOI DIFF bath （ 1 ： 136 ）
Diluent WBCbath （ 1 ： 417.7 ） WBCbath （ 1 ： 500 ） WBC/HGB
RBC bath （ 1 ： 20000 ） RBC bath/PLT
LH
Diluent

PD Mode
Flow cell
0.15ml
LEOII
DIFF
36°C
LEOI DIFF bath （ 1 ： 347 ）
Diluent WBC bath （ 1 ： 625 ） WBC bath （ 1 ： 750 ） WBC/HGB
RBC bath （ 1 ： 26040 ） RBC bath/PLT
Blood: 20ul
Diluent: 180ul
LH
Diluent

Aspirate sample and read HGB blank (0-6s)
Sample, vacuum, pressure prepare. Diff counting begin (6-25s)
Diff counting, WBC/Baso counting, RBC/PLT counting (25-42s)
Read HGB sample (43s)
Cleaning (43-59s)
Other Sequences

Start up Initialize liquid parts
Test vacuum
Test pressure
Test WBC volumetric tube filter
Test RBC volumetric tube filter
2. Shut down abnormally: 3. After pack-up:

1. Clean
Clean twice First prime, then clean
Background test

Shut down Pack-up
Clean Remove all the reagents
Use Cleanser Empty the tubing

to maitain
Connect diluent tube to distilled water
Clean the tubing via distilled water
Empty the tubing

Cleanser maintenance

Thanks!

BC5300 Hardware System
International Service Department

DIFF bath assembly
DIFF BATH

WBC/BASO and RBC assembly
WBC
RBC

Pump assembly

Pressure and vacuum chamber
Pressure chamber Vacuum chamber

Valve assembly 1

Valve assembly 2

Valve assembly 3

Thanks!

Technical Data BC-5300 - Five Part Diff-NA

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Optical System

International Service Department

Version: 1.0, Update Date: 2008-5-29

Version: 1.0, Update Date: 2008-5-29

Version: 1.0, Update Date: 2008-5-29

Normal flow Sheath flow

Version: 1.0, Update Date: 2008-5-29

• Sample flow width 20-30um.

Version: 1.0, Update Date: 2008-5-29

Version: 1.0, Update Date: 2008-5-29

Version: 1.0, Update Date: 2008-5-29

 For different cells of same size

Version: 1.0, Update Date: 2008-5-29

It is related to cellular contents (granularity, nuclear content, and so

 Flow cytometry + Laser Scatter

Version: 1.0, Update Date: 2008-5-29

Version: 1.0, Update Date: 2008-5-29

Lym Gran Mon

Eos Neu Bas

Dilute ratio 1:135(348)

RBC Lymphocyte Monocyte Basophil Eosinophil Neutrophil

Version: 1.0, Update Date: 2008-5-29

... . .. . ... . .. . ... . .. . Mon

Version: 1.0, Update Date: 2008-5-29

Version: 1.0, Update Date: 2008-5-29

Dilute ratio 1:500(753)

Version: 1.0, Update Date: 2008-5-29

Baso Lym, Mon, Neu, Eos

Dilute ratio 1:500(753)

Version: 1.0, Update Date: 2008-5-29

... . .. . .... .. Mon

Version: 1.0, Update Date: 2008-5-29

RBC Lymphocyte Monocyte Basophil Eosinophil Neutrophil

Version: 1.0, Update Date: 2008-5-29

Version: 1.0, Update Date: 2008-5-29

Note: MAS=SS LAS=FS

Version: 1.0, Update Date: 2008-5-29

Back Lens Assembly Splitter

■ Function: generate facula to irradiate the blood

Version: 1.0, Update Date: 2008-5-29

Version: 1.0, Update Date: 2008-5-29

Semiconductor laser technology

Version: 1.0, Update Date: 2008-5-29

Version: 1.0, Update Date: 2008-5-29

Version: 1.0, Update Date: 2008-5-29

Version: 1.0, Update Date: 2008-5-29

Version: 1.0, Update Date: 2008-5-29

Version: 1.0, Update Date: 2008-5-29

Version: 1.0, Update Date: 2008-5-29

Version: 1.0, Update Date: 2008-5-29

Version: 1.0, Update Date: 2008-5-29

• Remove the stray light and convenient to focalize.

Version: 1.0, Update Date: 2008-5-29

Version: 1.0, Update Date: 2008-5-29

Version: 1.0, Update Date: 2008-5-29

Version: 1.0, Update Date: 2008-5-29

SS 0.1Max ≤18 ≤15

Version: 1.0, Update Date: 2008-5-29

Version: 1.0, Update Date: 2008-5-29

4ul drain out

40uL: DIFF bath

Whole blood mode-20uL Pre-diluted mode