Automated cell counters-

THE BASICs
Pankhi Dutta MD DM (haematopath)
Consultant haematopathologist
SevenHills Hospital, Mumbai.
Introduction

Automated cell counterbackbone o! the

haemat lab

"allace Coulter in #$%& ' impedance

method

(arious technologies toda)

More accurate, more precise reports at a

!aster rate

*asic C*C + ne,er parameters

-nherent technological limitations

Basic parameters 3 part
counter

Haemoglobin

.*C, "*C, P/0 count

.ed cell indices

.D"

1part di!!erential

Histograms
NO. 4
DATUM: 9/10/95 15:11
MODE: VOLLBLUT
WBC 5,8 x
103/l
RBC 4,84
x106/l
HB 13,! "/#l
HCT 4$,0 %
MCV 86,8 &l
MCH $8,3 '"
MCHC 3$,6 "/#l
(LT $5! x103/l
L)M(H% 31,$ %
M*D% 6,8 %
NEUT% 6$,0 %
L)M(H+ 1,8 x103/l
M*D+ 0,4 x103/l
NEUT+ 3,6 x103/l
$50
RBC
RDW,-D 40,0 &l
40
PLT
(DW 13,1 &l
M(V 10,4 &l
(,LCR $8,1 %
WBC
300
3-part differential
analyser
0,o chambers

Hb + "*Cs

.*Cs + P/0s
Vacuum
Blood cell
DC supply
Registor
(constant current)
Internal electrode External electrode
Aperture
Transducer chamber
Blood cell suspension
(232& 1part Di!! technolog)
aemoglobin molecule
RBC
Ammonium
salts

!e"# !e"#

RBC

!e"# !e"#

1. Lysis of RBC
!e"# !e"# !e"# !e"#
Haemoglobin estimation
(232& 1part Di!! technolog)
aemoglobin molecule
RBC

!e"# !e"#

RBC

!e"# !e"#
!e"# !e"# !e"# !e"#

2. Change of conformity
(232& 1part Di!! technolog)
aemoglobin molecule
$ethemoglobin%complex

&table coloumetric complex ' directly proportional to b

Absorbance o( solution is measured against standard

!e)# !e)#

!e"# !e"#

*"

3. Oxidation
!e"# !e"# !e"# !e"#
DC detection method
Particle counting

DC direct current
impedance principle
volumetric measurement

"*C count and 1part di!!erential

.*C count

P/0 count
(232& 1part Di!! technolog)
DC Detection Method
(232& 1part Di!! technolog)
external
electrode
internal
electrode
aperture
+acuum
4 5 . 6 -
-mpulse
Impedance rinciple
Ext er nal
Elect r ode
I nt er nal
Elect r ode
Aper t ur e
V , R x C
V , Volt age
C , Cur r ent
R , Resist ance
Impedance rinciple
V , R x C
V , Volt age
C , Cur r ent
R , Resist ance
Ext er nal
Elect r ode
I nt er nal
Elect r ode
Aper t ur e
ro!lems- recirculation and
coincidence
A
B
C
pulse A pulse B pulse C
aperture
cells
(232& 1part Di!! technolog)
Samples are passing through the centre o!
the aperture ,ith sheath !lo, solution
for RBC & PLT
Hydrodynamic "ocusin#
7 .ecirculation and coincidence are prevented
7 8nhanced linearit) 9 accurac)
(232& 1part Di!! technolog)
- " ) . / 0 1 2 3 -4 -- -" -) -.
t
i
m
e
pulse height
From pulse to histogram: pulse
diagram
DC Detection Method
(232& 1part Di!! technolog)
- " ) . / 0 1 2 3 -4---"-)-.
Histogram
-4
"4
)4
- " ) . / 0 1 2 3 -4---"-)-.
Cumulative
Distribution
Curve
4 1 0 0 0 1 2 3 4 5 3 2 1 4
c
e
l
l
s
- " ) . / 0 1 2 3 -4---"-)-.
DC Detection Method
NO. 4
DATUM: 9/10/95 15:11
MODE: VOLLBLUT
WBC 5,8 x 103/l
RBC 4,84 x106/l
HB 13,! "/#l
HCT 4$,0 %
MCV 86,8 &l
MCH $8,3 '"
MCHC 3$,6 "/#l
(LT $5! x103/l
L)M(H% 31,$ %
M*D% 6,8 %
NEUT% 6$,0 %
L)M(H+ 1,8 x103/l
M*D+ 0,4 x103/l
NEUT+ 3,6 x103/l
$50
RBC
RDW,-D 40,0 &l
40
PLT
(DW 13,1 &l
M(V 10,4 &l
(,LCR $8,1 %
WBC
300
(232& 1part Di!! technolog)
"/%1/ ( l "44%"/4 ( l
Erythrocyte (RBC) istogram
u
.*C detection: bet,een ;% and ;%2 !/
u
Distribution curves are separated b) !le6ible
discriminators: ./ 9 .4
./
.4
.*C
P/0
(232& 1part Di!! technolog)
"/%1/ ( l "44%"/4 ( l
u
0he histogram curve should start and end at the base
line ,ithin the discriminators
./
.4
.*C
P/0
Erythrocyte (RBC) istogram
(232& 1part Di!! technolog)
"/%1/ ( l "44%"/4 ( l
u
-n case o! abnormal histogram curves the !lag
messages: ./< .4 or MP are generated and
results must be checked
u
./ : Abnormal height at lo,er discriminator
u
.4 : Abnormal height at upper discriminator
u
MP : (Multi Peak) .*C Anisoc)tosis
./
.4
.*C
P/0
!xam"#e$
RL f#ag message
-445
"45
A!normal Erythrocyte (RBC) istogram
(232& 1part Di!! technolog)
"%0 ( l -"%)4 ( l
( ixed at
-" ( l
P/
P4
P/0
.*C
-445
"45
u
P/0 detection: bet,een ; and 12 !/
u
=i6ed discriminator at #; !/
latelet (P#t) istogram
(232& 1part Di!! technolog)
"%0 ( l -"%)4 ( l
P/ P4
P/0
.*C
-445
"45
u
-n case o! abnormal histogram curves the !lag messages:
P/< P4 or MP are generated and results must be checked
u
P/ : Abnormal height at /o,er discriminator
u
P4 : Abnormal height at 4pper discriminator
u
MP : (Multi Peak) Platelet Anisoc)tosis
!xam"#e$
a%norma# PLT c&r'e
P( message
A!normal latelet (P#t) istogram
6ysing reaction to the 7BCs
&tructure o( 7B&
$itochondria
8ucleus
8ucleolus
Cell membrane
Ribosome
Cytoplasm
6ysing reaction on the 7BC
$eu%ocyte ()BC) istogram
Be(ore lysing reaction
0 $ 4 6 8 10 1$ 14 16 18 $0 $$
8eutrophile
Basophile
Eosinophile
$onocyte
6ymphocyte
Cell si9e in :m
-4 % -/
3 % -.
-- % -0
-" % "4
1 % -"
6ysing reaction and 7BC
A(ter lysing reaction
4 /4 -44 -/4 "44 "/4 )44
6ymphocyte
$onocyten
Basophile
Eosinophile
8eutrophile
)4 % 24
04 % -"4
14 % -)4
24 % -.4
-"4 % "/4
Cell +olume in (l
6ymphocyte
$onocyte
Basophile
Eosinophile
8eutrophile
$eu%ocyte ()BC) istogram
(232& 1part Di!! technolog)
"%0 ( l -"%)4 ( l
( ixed at
-" ( l
"/ "4
-445
"45
u
"*C detection: bet,een 12 and 122 !/
u
/eukoc)tes are separated in 1 parts:
l)mphoc)tes, mi6ed cells (mono, eo, baso)
and neutrophils b) discriminators: 0#, 0;
0# 0;
$eu%ocyte ()BC) istogram
(232& 1part Di!! technolog)
;)4 ( l %)44 ( l
"/ "4
-445
"45
u
0he histogram curve should start ,ithin the lo,er and
upper discriminator at the base line
u
Abnormal curves are !lagged ,ith "/, "4, 0#, 0;, =#,
=; 7 results must be checked
0# 0;
!xam"#e$
a%norma# )BC c&r'e
)L message
in case of
Lyse resistant RBC
A!normal $eu%ocyte ()BC) istogram
a!out 3-part differential
counters
>48S0-?@SA
&-part differential
counters
'arious technolo#ies (-
v
=luorescence !lo,c)tometr)
v
(olume Conductivit) Scatter
v
Pero6idase staining
')$*ME MEAS*+EME,T
(CS utilises the
Coulter Principle o!
counting and siBing to
measure the volume o!
the cell b) using Direct
Current (DC) across the
t,o electrode in a !lo,
cell.
Bec%man Coulter
C),D*CTI'IT- MEAS*+EME,T
Cell e6posed to .=,
the .= energ)
penetrates into cell
and reveal in!ormation
about its siBe and
internal structure.
SCATTE+ MEAS*+EME,T
As cells are pass in
single stream (!lo, cell)
the) are struck b) laser
strike ,hich gets
scattered.
0he light scatter at
angles bet,een #2 and 32
deg is used b) (CS
instruments.
The scattered light gives information about cell surface and
granularity
3D Data Analysis
L./'01
M2321
B4121
NRBC1
E21
N5671
AD'IA TECH,)$).-
"*C and Di!!erential
Pero6idase Channel Stain Cells "ith
Pero6idase
:8osinophils Strong Staining
:@eutrophils Medium Staining
:Monoc)tes "eak Staining
:/)mphoc)tes and *asophils
@o Staining
:/arge 4nstained Cells (/4C)
@o staining
Also Measure Cell SiBe 4sing /o, Angle
ScatterPlot ;D
Scattergram 0o Cive D Part Di!!erential
E21832'08l1
N56792'08l1
M232:.751
LUC
L./'02:.751
;
B412'08l1
Pero6 Activit)
(o
lu
m
e
0he AD(-A "*C di!!erential is calculated
!rom a 1 step process.

Cells are stained b) pero6idase

reagent and anal)Bed !or siBe and
pero6idase stain intensit).

Cell speci!ic l)sis reagents are used

to separate basophils !rom all other
,hite cells.

*asos are subtracted !rom the

l)mphEbaso cluster in the pero6
channel to calculate the l)mphs.
AD'IA TECH,)$).-
Sysme/ 0-class analy1ers-
"luorescence flo2
cytometry
"luorescence flo2 cytometry-
3li#ht scatter and fluorescent
dyes4
D$
Differential-
"Sc 5s SSc 3!aso channel4
S"$ 5s
SSc
3diff
channel
4
ACAS 6 Centroids
&&C
&!6
<host
8eut # Ba
$ono
6ymph
Eo
-= The ( ir st cent r oids ar e pr o+ided>
The starting position o centroids has been
determined rom thousands o samples!
These values are stored in the instrument
and are used as the starting position or
cluster anal"sis!
ACAS 6 Mahalano!is
Distance
;. Cluster anal)sis o! scattergram
-! a cell is detected, distances bet,een this
signal and the given centroids are calculated
(Mahalanobis distance). 0his distance reveals
to ,hich given cell population the signal
belongs.
&&C
&!6
<host
8eut # Ba
$ono
6ymph
Eo
-= calculated centroids
$ahalanobis%
Distance
Differential fluorescent
stainin#- Immature
#ranulocytes3Sysme/4
Diff scatter#ram( I. positi5e 5s I.
ne#ati5e
IG MASTER
+eticulocyte parameters 3+ET
channel4

Separate channel

Pol)methine d)e
stains @.A. in "*Cs,
n.*Cs , retics 9
platelets.

SiBe vs !luorescence

.etic count
+etic channel

.eticuloc)te count

.eticuloc)te !ractions (/=., M.=, H.=)

-mmature reticuloc)te !raction

.etHe (reticuloc)te Hb content)

Platelet '? (!luorescent platelets)

=ragmented red cells (=.C)

An example of efficient multitasking!!
,e2 haematolo#ical parameter
can predict iron deficiency
2here classical serum tests
fail

.eticuloc)te Haemoglobin

8Fuivalent .etHe

CHr Siemens

(=DA approved)
Ret-He

R57,H5 , H< :237537 5=68>4l537 2&

9578:6l2:.751

U3871 2& ?'"@A329/4l 943"5, $8 B 35 '"C

M2387291 17475 2& 8923 16''l. #6983" 705

:26915 2& 59.7092'285181, '92>8#51
83&29/47823 23 availability of functional
iron.

Can classify hypochromic anemia,

classical vs functional ID, helps to
select optimal therapy & to monitor
response to EPO and iron treatment.
Spurious platelet counts-
Siegenthaler & Spertini, NEJM May
2006

DG )r old M ,ith
severe burns

Automated C*C Hct

13H, MC($#!l, .D"
#%.GH, "*C3,D22Eul,
P/0 count ;3D222Eul

PS '
microspheroc)tes
and spheroc)tes

Manual platelet
count G%,222Eul
)5ercomin# the pro!lems
2ith impedance countin#

Manual method haemoc)tometer ,ith

phase contrast
(0ime consuming, laborious,
operator dependenc) is more)

=lo,c)tometric method using .*CEP/0 ratio

(anti CDD#, anti CD&#) Am J Clin Path 2001
(86pensive, reFuires a !lo,c)tometer and
e6perience ,ith =CM)
)ptical (fluorescent)
platelets 3#ood correlation
2ith reference methods4
S)sme6 !luorescent P/0? results are unmatched b) ordinar)
platelet technologies o! other anal)Bers
Fluorescent P!
(platelet"#)
*icrocytic RBC
Giant PLT
$T a!n7 Distri!ution #iant
throm!ocytes
I" Immature latelet
"raction
-mmature P/0 are identi!ied b) its increase in
f#&orescence (more .@A), +,C is a#so higher.
IPF
eripheral smear
e/amination still
re8uired99
Throm!ocytopenia
latelet clump3EDTA
induced4
latelet count after
collection in citrate 99
Criteria for smear re5ie2

Smear revie, increases manual ,ork,

reduces 0A0 9 productivit)

@eed to reduce smear revie, rate ,ithout

risk o! missing an)thing signi!icant

Di!!erent labs ' di!!erent criteria

Criteria depend upon patient population,

t)pe o! anal)ser in use, etc.
Consensus rules for smear
re5ie2

-nternational consensus group

!or hematolog) revie, ' -S/H,
;22; ( Dr. *erend Hou,en)

/aid do,n rules !or action

!ollo,ing automated C*C
including smear revie,

.ules tested in #% labs (#1,;$G

samples)

Data anal)sed, rules re!ined,

D1 rules laid do,n

Cuidelines !or individual

laboratories
+e5ie2 ( Criteria for
automated CBC : ;BC diff
analysis
E/amples of some consensus
rules 3a$ %ae&atol, 200'4
.ule no Parameter Primar) AndEor Action
# neonate #st sample Slide revie,
#2 MC( I3%!l or J#2%!l Specimen
I;Dhrs old
Slide revie,
#% .D" J;; #st time Slide revie,
#& @o "*C
di!!Eincomplete
Manual di!! 9
slide revie,
3 Platelet I#22 or J#222 #st time Slide revie,
!lagged normal Anal)ser
.outine
technician
A Abnormal .*Cs
A At)pical mononuclear
"*Cs
D
Common .*C abnormalit)
D
Cranuloc)te le!t shi!t
D
At)ipcal (variant) l)mphoc)tes
D
normoblasts
Sr. technician
A *lasts
A ?rganisms
D
M)eloc)tes
D
Plasma cells
D
Dohle bodies
D
0argets
D
Auer rods
Ph)sician
Diagnostic
cells
.eport .eport .eport .eport
86aminer
Di!!erentiation
Hierarchial blood !ilm evaluation
<*I=-
Fragments ?
Eosinophilia
Iron Deficiency Anemia
hoto of slide
Lymphocytosis
Summary

Automated cell countersbackbone o! the

diagnostic laborator)

=ast, accurate, precise

-mpedance and various other technologies

All have various limitations

Accurate in!ormation on the technologies

help!ul to recogniBe problematic areas

Maintenance, calibration, >C procedures

Slides still need to be revie,ed (criteria)

=inall)KKK
IT IS THE MAN BEHIND THE MACHINE WHO
MATTERS MOST!!
THANK YOU!!