Pentra 60 & 80 5DIFF Technology

1
12O P60 5DIFF BASO LMNE EN Ind A

Restricted Diffusion
© 2007 HORIBA, Ltd. All rights reserved.
WBC/ Baso and LMNE
on P 60 & P 80
HORIBA Medical Training Center

Montpellier
2014

WBC/Baso and LMNE Principles
 Contents:
MDSS : distribution
WBC/Baso circuit
WBC/Baso dilution
LMNE dilution
LMNE transfer dilution
LMNE injection
WBC formula
Absorbance measurement
Alarms
3
MDSS : Distribution
Sampling Distribution Mixing
4
MDSS : Distribution
WBC/BASO
Blood 10µL
RBC/PLT Basolyse II 2mL
Final Dil. : 1/200
1st Dil. 42,5µL
LMNE Diluent 2.5mL
Blood 25µL Final Dil. : 1/10000
Eosinofix 1mL
1stdilution
Diluent 1mL
HGB Final Dil.: 1/80
Rinsing
Blood 10µL
Blood 3µL
Diluent 2.1mL
Blood 5µL
Lyse 0.4mL
Final Dil. : 1/250
5
WBC/Baso circuit
WBC/Baso
chamber
6
WBC/baso dilution
The electronic measurement
principle is the same as the
RBC/PLT.
• An aliquot of 10 µl of blood Sampling needle
is pourred into the WBC/Baso
chamber with a synchronized
flow of 2 ml of Basolyse II.
• Reaction temperature : 35°Celsius
• Incubation time : 9 sec
The Basolyse II lyses RBC, lyses PLT,

lyses WBC which release their nucleus, P R L M N E B
excepted for the basophils population.
7
WBC counting
WBC / Baso
Chamber
Vacuum WBC pulses
Count 1 (5 seconds) Count 2 (5 seconds)
2 counts of 5
seconds
𝐖𝐁𝐂 =
Aperture (𝑾𝑩𝑪𝟏+𝑾𝑩𝑪𝟐)
𝟐
×𝑲wbc×𝒌
WBC count (80µm)
Kwbc = WBC calibration coefficient
Impedance K = coïncidence coefficient
principle
8
WBC Formula
Then, using WBC/Baso chamber, trought an aperture of 80µm, count is done
and volumetric pulses are obtained. These pulses are then displayed on a
volumetric histogram :
Number
Volume
WBC nuclei Baso
If we get a total of 300 pulses, with 297 between 1 & 2 thresholds,

and 3 between 2 & 3 threshold, we have 3/300 = 1% of basophils.
9
LMNE Dilution
• The blood aliquot (25µl) is poured
into a synchronised tangential flow
of 1ml of Eosinofix into the LMNE Sampling needle
chamber
• Incubation time : during 12

secondes at 35° Celsius.
During incubation, the Eosinofix

1 lyses the RBC,
2 gives a specific black coloration
to Eosinophils
3 protect the others WBC volume.
P R L M N E B
Then 1 ml of diluent is poured
into LMNE chamber, dilution is 1/80
10
LMNE transfer dilution principle
LMNE chamber
LMNE injection syringes

11
LMNE injection principle
Flowcell
Back to LMNE chamber
1st sheath with

diluent
2nd sheath with diluent
LMNE syringes
12
WBC Formula
Volumetric absorbance
measurement measurement
The percentage of
the white blood cell
populations is
obtained by flow
2nd sheath with diluent
cytometry.
Injection speed : 12 km/h

1st sheath with diluent
Injected volume : 72 μL
13
WBC Formula
•Constant speed, it is possible
with the transfer time to check
that the volumetric pulse and transfer time
the absorbance pulse belong to 200 µs
the same cell (correlation
notion)
• In case of large quantity of absorbance

WBC (ex: hyperleukocytosis, volume
leukemia), the system will
take into account the
volumetric and absorbance
informations of the current
cell prior to perform the
measurement for a next one
14
Absorbance measurement
Crossing light
Diffracted light
 Emitted light
Transmitted light Absorbed light
Absorbance All of the cell characteristics

Emitted light will be taken into account.
Transmitted light
15
WBC Formula
Cell Aspect Volume Lost light
(Absorbance + diffraction)
Lymphocytes Small A little
Monocytes Big A little +
Neutrophiles Medium Medium
Eosinophiles Bigger due to the Big (Reagent Action)

Reagent action
16
WBC Formula
WBC localisation on matrix
Eosinophils
Neutrophils
Lymphocytes
Large immature cells
Atypical Lymphocytes Monocytes
17
Raw Values
The raw counts are printed at the bottom of the Report printout.
Avalaible on printout only
18
Raw Values
If activated, the raw counts are printed at the bottom of the Report printout.
19
Raw Values (CBC)
WBC = (WBC1+WBC2)/2 x Kwc x k

Example :
Kwc = calibration coefficient of WBC
WBC = 6,6 103/mm3
K = coïncidence coefficient
20
Raw Values (DIFF)
LIC = RN + RM + C127
DIFF# = LYM + MON +

NEU + EOS
DIFF#
CO % ≈ x 100
RA #
21
Alarms
WBC / Baso Alarms :
L1 alarm can be triggered out either on DIF or CBC mode.
-> WBC result will be flagged with a « ! » (suspicion point)
Values for L1
if 3 % or 200 #
Suspected abnormalities:
PLT aggregates
NRBCs
Small lymphocytes
22
Alarms
5 DIFF Alarms and others populations :
2% 80#
3% 100#
5% 55#
23
Alarms
1.8% 60#
0.5% 40#
0.7% 60#
24
Alarms
► Separation alarms:
2% 120#
1.2% 100#
1.1% 30#
25
Alarms
►Other populations:
+ Channel 127
3% 0.3#
2.5% 0.25#
26
Alarms
NO:
Lymphocytes – Monocytes – Neutrophils - Eosinophils
Platelets aggregates NE:
NRBC’s Young eosinophils
Erythrocyte membrane Hypersegmented
Large number of platelets neutrophils
Pollution Imature cells
LN: LIC:
Neutrophil destruction (old blood) Large monocytes
Contamination Large neutrophils
Stroma Myelocytes..
Platelet aggregates
NL: RN:
Slighty neutrophils Large neutrophils
Segmented lymphocytes Imature cells
Activated lymphocytes (metamyelocytes,
Hyposegmented lymphocytes promyelocytes..)
RM:
LL / LL1 Large monocytes
Small lymphocytes Myelocytes,
Platelet aggregates Promyelocytes
NRBC’s Large blasts
Resistant RBC
MN:
ALY: Hyperbaosophilic
Large lymphocytes monocytes
Plasmocytes Young neutrophils
27
Alarms
Other alarms:
• LMNE+; LMNE- : if the difference in + or in – between the WBC/Baso channel

and the LMNE channel are higher than the defined conditions.
• BASO+ : if the Baso% found in the WBC/Baso is greater than 50 %

and BASO result will not be reported « --.-- ».
• CO : if correlation between resistive and optical measurements in the LMNE

channel is less than 50 %
-> LMNE results will not be reported « --.-- »
28
Any questions ?
29

Pentra 60 &amp; 80 5DIFF Technology

Uploaded by

Document Information

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Pentra 60 &amp; 80 5DIFF Technology

Uploaded by

Copyright:

Available Formats

1

12O P60 5DIFF BASO LMNE EN Ind A

HORIBA Medical Training Center

© 2007 HORIBA, Ltd. All rights reserved.

Sampling Distribution Mixing

• Incubation time : 9 sec

The Basolyse II lyses RBC, lyses PLT,

Count 1 (5 seconds) Count 2 (5 seconds)

WBC nuclei Baso

If we get a total of 300 pulses, with 297 between 1 & 2 thresholds,

• Incubation time : during 12

During incubation, the Eosinofix

LMNE injection syringes

Back to LMNE chamber

1st sheath with

Injection speed : 12 km/h

• In case of large quantity of absorbance

Absorbance All of the cell characteristics

Lymphocytes Small A little

Monocytes Big A little +

Neutrophiles Medium Medium

Eosinophiles Bigger due to the Big (Reagent Action)

Atypical Lymphocytes Monocytes

WBC = (WBC1+WBC2)/2 x Kwc x k

DIFF# = LYM + MON +

• LMNE+; LMNE- : if the difference in + or in – between the WBC/Baso channel

• BASO+ : if the Baso% found in the WBC/Baso is greater than 50 %

• CO : if correlation between resistive and optical measurements in the LMNE

You might also like

Pentra 60 & 80 5DIFF Technology

Pentra 60 & 80 5DIFF Technology