BC-5600 Optical System

BC-5600 Optical System
Hematology Global Technical Support Department

Service@mindray.com
Version ： 1.0
NO.:MXQ-12023-BC-5600
© 2011 Mindray Confidential

OTY-12002 （ 3.0 ）
Optical System
 5 Diff Principle
Optical System Structure
Optical System Adjustment
Troubleshooting
2 © 2011 Mindray Confidential

5 Diff Principle

Blood Cells
 Blood consists of 40-50% blood cell and 50-60% plasma.

 Three main parts of blood cell:
white blood cell, red blood cell and platelet.

Blood Cells
Neutro
Eos RBC
Mono
PLT
Baso
Lymph

What is 3-part diff?
Normal size sequence
After Lysing
What is 5-part diff?
Five groups of Leukocyte (stained)

Lymphocyte 25 ~ 30%
Monocyte 3 ～8%
Neutrophil 50 ~70%
Basophil 0 ～1%
Eosinophil 0.5 ～ 3%

Similarity and difference
Small granules; Multi-lobed

nucleus
Large dark colored

Large round Nucleus fills most of granules; purple lobed
the cell nucleus
Large red-orange granules;

purple bi-lobed nucleus
More cytoplasm than Lymph
Simple Structure & Different Size Complex Structure & Mid-sized

How to identify the five parts
Hydrodynamic Focusing
Multi-angle Laser Scatter
Blood Cell Morphological Treatment

Flow Mode of Cell Suspension
Sheath flow Normal flow
3-diff analyzer

Hydrodynamic Focusing
• Sheath flow surrounds and forces blood cells suspended in dilution to pass through the
center of flow cell one by one.
• Blood cells pass through the Laser spot of flow cell with high speed.
• Sample flow width 20-30um.
20~30 um
Multi-angle Laser Scatter
High Angle Scatter 8~20

5
reflects granularity and
complexity
2
3 4
Low Angle Scatter 2~5
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 (2~5) 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

High Angle Scatter (MAS)
The high 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?

Blood
LEO(I) RBC
LEO(II)
Lymph Eos Neu Mono

DIFF channel
Morphological treatment
SIZING STAIN
Eos
Neu
... . .. . ... . .. . ... . .. . Mono

. .. . .. . .. .
. .
Lymph

Blood
LBA RBC
Baso Lymph, Mono, Neu, Eos

BASO channel
Morphological treatment
SIZING
Eos
Neu
Baso
... . .. . .... ... Mono

. .. ..
.
Lymph

Scatter gram
Horizontal axis: High angle scatter signal reflects the internal complexity of cell.
Vertical axis: Low angle scatter signal reflects the volume of cell.
Neu
Baso
Mono
Lymph
Total
WBC
Eos

DIFF
Large size
Neutro is complex and big
Mono has larger size than

Lymph.
Lymph’s internal structure is

much simpler than Eos’s.
Small size
Simple structure Complex structure

BASO
Large size
Baso keeps the same size.
Lymph
Neutro
Eos
Mono
Small size Shrink greatly.
Simple structure Complex structure

How to get the 5-part results?
First count Diff channel, get

 Lymph%, Mon%, Eos%, Neu%
Then count Baso channel, get

 WBC, Baso#, Baso%
Then get
 Lymph#, Mon#, Eos#, Neu#



board
Laser control
MAS PD Assembly
Assembly
LAS PD
LAS Beam Stop
MAS Beam Stop
Flow Cell
Assembly
Back Lens Assembly Splitter
Forward Optical Assembly Backward Optical System

Forward Optical Assembly
 Generate laser spot to irradiate the blood cell.

Forward Optical Assembly

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

Flow Cell Assembly
 Generate stable sample flow; make blood cell in the sample flow pass trough laser spot
(irradiation spot) one by one.

Flow Cell Assembly

Backward Optical System
 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

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.
MAS Stop Assembly
LAS Stop Assembly

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

Optical System Adjustment

Adjust Alignment
Use AllenKey to adjust and ensure forward

assembly parallel with back lens Adjust to make filtered area
Press Back lens against
the pins
Forward Backward Splitter

assembly Lens
Low Angle collector ( 2-

5 degree)

Adjust Alignment
1. Install the back lens assembly. Rotate the forward assembly to let it be parallel with
back lens assembly
Pitch Pin
1
Adjust Alignment
2. Adjust flow cell assembly (1): Rotate the flow cell to let it be parallel with the forward
assembly.

Adjust Alignment
3. Install splitter and adjust beam stop
Symmetric
3
3

Adjust Alignment
4. Move back lens assembly to let the light spot in the middle of the beam stop

Adjust Alignment
5. Move flow cell into optical path, until you see two clear lines and make them in the
middle as below
Flow cell edge

Adjust Alignment
6. Adjust direct light beam stop

Adjust Alignment
7. Adjust PD assembly
High angle scatter
Low angle High angle

Adjust Focus Position
Tools:
FLUKE124

Test points:
Pre-amplify board Signal processing board
LAS TP43---LASIN
MAS TP44---MASIN

1. Adjust forward light axis position

Standard: Let LAS pulse be biggest, width smallest(1.3us), and stable.
Oscilloscope settings ： AC Coupler ， Voltage 50mv ， Time 500ns

2. Adjust back light axis position

Standard: Let MAS pulse be largest.
Oscilloscope settings ： AC Coupler , Voltage 100mv ， Time 500ns

FAQ
 What is the key point of each step?

Practice
 Let’s practice by Group!

Summary
 Group 1
 Group 2

Troubleshooting
 Understand the Normal Scattergram

 Abnormal Scattergram
 Troubleshooting method in summary

Understand the Normal Scattergram

Normal scattergram of sample

DIFF scattergram:
1. The 4 parts can be differentiated
Neu
separately.
2. The 4 parts of DIFF channel should be 3/4 or 4/5 of DIFF screen.
Mono
Eos
Lymph Baso
4 Parts

BASO scattergram:
1. The 4 parts except BASO
Neu
should be close to the lower left corner.
2. The profile of lower part should be smooth, not cut by a straight line.
Mono
Eos
Lymph Baso
RBC Ghost 4 Parts

Normal scattergram of standard particle
DIFF BASO

Normal scattergram of standard particle
 7um standard particle: use to check the LAS/MAS 0.1max width

At the "Count" screen, run the 7um standard particles in the OV-WB-CBC+5DIFF mode, and then go to the "Review"
screen. Select the analysis record and click "OpAdjust". Click "Calculate", and check the particle with larger "LAS
0.1max Number“.
The requirements: LAS

0.1max Width≤9;
MAS 0.1max Width≤13

Abnormal Scattergram

Scattergram of abnormal sample
Leucopoenia Eosinophilia
Lymphocytosis Neutrophilia Monocytosis

Scattergram of abnormal sample
ATL LIC
Left shift Lyse resistant

Scattergram caused by Hardware failure
 Confirm the Hardware

Laser light exists?
Cable connecting PD Board and Analog Board?
PD Board?
Analog Board?

Scattergram caused by Dirty/Clog tubing

Flow cell dirty
 Clean the flow cell

Flow cell dirty
 Clean the flow cell

Scattergram caused by improper Gain

Confirm the Gain
 Confirm the optical system

Calibrator: used to check the LAS/MAS CG position
At the "Count" screen, select the mode "OV-WB" and "CBC+5DIFF", and then run the calibrator three times
(recommended). Select the first analysis record of calibrator in the “Review”, and click "OpAdjust".

Confirm the Gain

Select "Calibrator" mode and click "Calculate". Record the "LAS/MAS CG Position" of the particle with larger "LAS 0.1max Number",
and then check the "LAS/MAS CG Position" of the other 2 analyses .

Confirm the Gain

Calculate the mean values of "LAS/MAS CG Position" of the 3 analyses, and make sure: LAS CG position mean within ±2.80 from the
target; MAS CG position mean within ±3.20 from the target.

Confirm the Gain

If the mean values are out of the range, enter the "LAS CG Target" and "MAS CG Target" and click ">>>" to get the gain values for the
1st calibrator analysis (note down the new gains).
Select the right Particle group

as the previous pages
If you set the new gains just

by 1 calibrator test, not three
times tests as recommended,
you could directly click this
button to apply the new gains

Confirm the Gain

Configure gain of the other 2 analyses in the same way. Calculate the mean values of the LAS gain and MAS gain of the 3 analyses as
the final gains. Click "Setup"→"Gain", and enter the final gains in the "WBC_LAS(DIFF)" and "WBC_MAS(DIFF)" fields. In the
"Corresponding Factor of DIFF and BASO" area, enter 1.87 in the LAS field and 1.48 in the MAS field.
After setting DIFF gains,

use the factors to set the
BASO gains automatically

Confirm the Gain

Control: If the calibrator is not available, use the normal control instead; the operation
is the same, while just having different target values.
The target value of LAS/MAS CG Position is different according to the lot no.. Please contact
Mindray service engineer to get the right one.
If the normal control is not available, use the normal fresh blood to adjust the gain
generally.

Confirm the Gain

 If scattergram is compressed too much, adjust the forward focus.

Confirm the Gain
• Confirm the optical system

Adjust the forward focus.
Forward optical
assembly First loosen these two screws
Flow cell
Then adjust this screw

Case study
After clean and

adjustment

Case study
After clean and

adjustment

Troubleshooting method in summary

Troubleshooting method in summary
 First,
 Check reagents, Clean the WBC counting channel with software
 Second,
 Identify the situation according to scattergram
 Hardware failed?
 Dirty/Clog?
 Improper gain?
 To be continued in the Liquid System

Version Information
Version Author Date Description
1.0 Eric cheng 2012.6.18 BC-5600 Optical System

Check Information
Version Reviewer Date Comments
1.0 Carl 2012.6.20 Initial Release

Thank you!

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Hematology Global Technical Support Department

© 2011 Mindray Confidential

2 © 2011 Mindray Confidential

© 2011 Mindray Confidential

 Blood consists of 40-50% blood cell and 50-60% plasma.

4 © 2011 Mindray Confidential

5 © 2011 Mindray Confidential

Normal size sequence

Five groups of Leukocyte (stained)

7 © 2011 Mindray Confidential

Small granules; Multi-lobed

Large dark colored

Large red-orange granules;

More cytoplasm than Lymph

Simple Structure & Different Size Complex Structure & Mid-sized

Multi-angle Laser Scatter

Blood Cell Morphological Treatment

9 © 2011 Mindray Confidential

Sheath flow Normal flow

10 © 2011 Mindray Confidential

• Sample flow width 20-30um.

High Angle Scatter 8~20

13 © 2011 Mindray Confidential

 For different cells of different size

14 © 2011 Mindray Confidential

15 © 2011 Mindray Confidential

 For different cells of same size

16 © 2011 Mindray Confidential

 Flow Cytometry + Laser Scatter

18 © 2011 Mindray Confidential

 Is Flow Cytometry + Laser Scatter enough to have 5-part?

19 © 2011 Mindray Confidential

Lymph Eos Neu Mono

20 © 2011 Mindray Confidential

... . .. . ... . .. . ... . .. . Mono

21 © 2011 Mindray Confidential

Baso Lymph, Mono, Neu, Eos

22 © 2011 Mindray Confidential

... . .. . .... ... Mono

23 © 2011 Mindray Confidential

24 © 2011 Mindray Confidential

Neutro is complex and big

Mono has larger size than

Lymph’s internal structure is

Simple structure Complex structure

Baso keeps the same size.

Simple structure Complex structure

First count Diff channel, get

Then count Baso channel, get

27 © 2011 Mindray Confidential

© 2011 Mindray Confidential

29 © 2011 Mindray Confidential

Back Lens Assembly Splitter

Forward Optical Assembly Backward Optical System

30 © 2011 Mindray Confidential

 Generate laser spot to irradiate the blood cell.

31 © 2011 Mindray Confidential

32 © 2011 Mindray Confidential

Semiconductor laser technology

33 © 2011 Mindray Confidential

34 © 2011 Mindray Confidential

35 © 2011 Mindray Confidential

36 © 2011 Mindray Confidential