Validation procedures for cell

analyzers
Dr Archana Vazifdar
Dept. of Hemato-Pathology,
Super Religare Laoratories Limited,
!umai
Principles of automation

Impedance " count and size cells y

change in resistance produced as they are
suspended in an electrically conducti#e
medium

Optical scatter- measures scatter

properties of cells y laser light

Single angle$ !ulti-angle scatter

R%& ' Platelets measured in one channel

R%& #olume ( )*-)+ fl

Platelet #olume ,-,* fl

H ' -%& measured in second channel

DL& in third channel

.nterpretation of data
/ormocytic
/ormochromic
RBC count
Spurious increase0
1
2iant PL3
1
High -%& counts 4(5*6
Spurious decrease0
1
&old $7arm agglutinins
1
Very small R%&
1
&ryogloulins
ADVIA 120
CELL-DYN
COULTER
Platelet count
Spurious increase0
1
R%&$ -%& fragments
1
&ryogloulins
1
Lipids
Spurious decrease0
1
Platelet clumps
1
2iant platelets
neutro
lympho
Baso,mono, eos,
blasts
WBC (FCM)
Normal WBC scatterplot
Normal WBC histogram
Impedance- VCS
Optical scatter: !"I#$%
DL& y Pero8idase method
Spurious increase
1
PL3 clumps ' large platelets
1
/ucleated red cells
1
Resistant R%&9s
Spurious decrease0
1
&lotted sample
1
:ragile cells- &LL
1
Lymphoid aggregates- ;3., %- cell /HL,
&!!L
1
Storage associated degeneration
:lags

A signal to the operator that the analyzed

sample may have a significant abnormality/ does
not meet acceptance criteria$ cannot e
displayed

&ause of errors0

Analyzer

Sample

Random run error

R%& flags
&uspect 'la(s
1
/9rc, R9rc, !icro R%&, R%& fragments,

interfere 7ith -%& ' platelet counts

1
H ' h errors
1
short sample, aged sample
!e'initi)e 'la(s
1
Anemia, anisocytosis, microcytosis,
macrocytosis, poi<ilocytosis
1
=rythrocytosis
FLAG:
Anemia, Microcytosis, anisocytosis
H >.5
R%& ).,
Left shift of curve:
Microcytosis
Iron Deficiency Anemia
thalassemia trait
Anemia of chronic diseases
&onclusion0
s*o Iron !e'icienc+ nemia
Ad#ise .ron studies
ACTION:
RBC indices
Mentzers inde !MC"#RBC$%
&'()
MI * &)+ BTT, , &)+ IDA
:lags:
1
Nr-c, Micro RBC# RBC
fra.ments
1
/iant 0lt
1
Throm-ocyto0enia
Lt of curve not touching
baseline:
Noise
1chistocytes 2# etremely
small r-c
/iant 0latelets
PL3 ?@*
!PV A.B
P&3 .?@>
PD- ?5
H +.@
&onclusion0
R%& count falsely C
Platelets falsely D 4mas<
t9penia6
,emol+tic anemia
Action0
1
R%& .ndices- !&V, RD-
1
PL3 Histogram- !PV ' PD-
1
Re#ie7 PS- R%& morphology
-PL3 count 4?**6
Bimodal peak:
Dimor0hic RBC 0o03lation
Transf3sed cells
Com-ined deficiency
Thera0e3tic res0onse in IDA
,-. /012 MC,. $1032
MC,C. 4$0$
:lags:
Dimor0hic RBC 0o03lation, anisocytosis
Action0
Re#ie7 PS to identify cause
50/ F,
Hb-8., !C"-#$, !CH- %5.&, RDW-26.8
Bloo' trans(usion
)ual/Combine' 'e(iciency
*5/F, +e,ere pallor
Hb-5.-, !C"-&.#, !CH- %.&, !CHC-$-.*, .)W-%*.5
/0C/1lt-Normal
+. Fe- %5
/2BC- -**
+. Fe saturtn- %0.8
+. B-%- -58
Right portion of curve extended:
RBC a..l3tination
Nr-cs
4e35ocytosis
:lags:
626 error, Nr-c, dimor0hic reds
Anemia, macrocytosis, anisocytosis
H'H

Sample related prolems- turidity-D H

Lipemia$ 3P/

&ryogloulins

Autoagglutination

Hemolysis 4in-#itro$#i#o6

Spurious C Hct

&lotted sample
&purious 5MC,C:
corrected
&onclusion0
:alse C R%&, Hct,
:alse D !&V, !&H ' !&H&
Cold a((lutinin disease
After 7arming in H,E ath
F )AG& for ?5 mins
Action:
Re7ie8 91: 4#: a..l3tination 7s nr-cs
+hort sample 3microtainer4
.epeat collection
Causes o( H5H mismatch6
1
partial sample aspiration/ improper mi7ing
1
Hb/ !C" measurement error/ ,ery lo8
1
High WBC counts 3inter(ere 8ith Hb
measurment4
1
Col' agglutinins
Platelets
Smallest guys largest culpritsHH

As platelet counts fall, reliaility of

analyzer decreases.

&on#entional methods are unale to

pro#ide consistently accurate results in
lo7er range

&linicians using thresholds of 5-?* I ?*B$l

must e a7are of the limitations in
precision and accuracy of cell counters
Linearity 0 ?*"?,*** I ?*B$l
&ommon platelets flags

PL3 &lumps

CPlt counts

.nterferences 7ith -%& Results 4D-%&

counts6

2iant platelets

Small platelets

P.&$PE& delta- difference ( ,*,***

3hromocytopenia- true$false
Increased small si6ed particles:
/oise, deris, lipids, acteria, fungi
J -is<ott Aldrich syndrome
&onclusion0
:alsely ele#ated platelet counts
Fla(s:
Small platelets
Deris$ noise
ction:
Re#ie7 PS for
platelet count
&onclusion0
:alsely DR%& count
:alsely D-%& count
:alsely C Plt count, D!PV
7iant platelets
Fla(s:
2iant platelets, platelet
clumps
&ellular interference
8on 'itted cur)e 9it:
increase in lar(e cells:
Large platelets, clumps
P.&$PE& delta
1
=8cessi#e noise
included in impedance
count
1
Deris, acteria, fungi
1
Plt clumps
1
2iant plt
@5$!
.2, %and, %lasts
Aty ly, Variant ly
!PE, non #iale -%&
/9R%&, rst R%&
Plt clump
Eutside Reportale Range
Leu<ocytosis, monocytosis, asophilia, eosinophilia
;nale to :ind &lear Separation et7een -%&
supopulations
-%& :lags
Shoulder on the left of curve:
Nrbc
Lyse resistant RBC
Platelet clumps/ Giant platelets
Fibrin
Impedance noise
Fla(s: .2, %lasts, eosinophilia,monocytosis, lymphopenia
&!L
Leu<ocytosis
3hromocytosis
Anemia
:lags0
Aty lymphocyte,
Variant lymphocyte
/on-#iale 7c
Leu<ocytosis
39penia
Acute Leu<emia
)>$:, <$c$o D!
:lag0 leu<ocytosis, n9rc, dimorphic reds
&onclusion0
,? nrc9s$?** 7c- corr -%&K ?A.)5
D! in sepsis 7ith li#er ascess
Plt ?**
VCS:
1
Luantitati#e
1
Eperator independent
1
Routinely a#ailale
1
.ne8pensi#e
./&R=AS= !=A/ /=;3REPH.L VEL;!= 4!/V6
D=&R=AS= !=A/ /=;3REPH.L S&A33=R 4!/S6 "
left shift
"
Lac;in( leu;oc+tosis or neutrop:ilia
/e7er Aspects0
V&S-/eutrophil population data
&u((esti)e o' acute -acterial sepsis
utomated malaria detection

M2old standardN - thic< ' thin smear

/eed for rapid, sensiti#e ' cost-effecti#e

screening techniOue

Hemazoin pigment
1
Acti#ation of neutrophils ' monocytes
1
.ncrease #olume heterogeneity 4anisocytosis6 of
monocytes ' lymphocytes, detected y V&S

PPositional parameters9, used as oQecti#e

criteria for detecting presence of plasmodium
Clin. 0ab. Haem., %&, $&#9$#% :utomate' 'etection o( malaria
Normal
Plasmodium falciparum
Monocytes
Reacti7e 4;
9arasitized RBC
Vol SD lymphocyte I SD !onocyte $ ?**
( ).A
:m ; Clin 1athol %00&<-%&6&--&8
Briggs et al / !:0:.2: )=/=C/2>N ?+2N@ "C+ /=CHN>0>@A
shoul'er

Specificity is B@R and sensiti#ity B>R

PPV is A*R and /PV BB.AR.

A flag indicating potential presence of

malaria is a #aluale diagnostic method for
detection of malaria and may ecome a
routine parameter in it9s diagnosis
Reticuloc+te Indices

most promising from a clinical #ie7point are the &Hr and

the !&Vr.

C,r:

directly reflects hemogloin synthesis in marro7, ' measures

iron a#ailaility.

C .DA ' %33 4independent of iron stores6

MC"r: 5rapidly follo7ing iron therapy

C 7ith the de#elopment of iron-deficiency

C in macrocytosis after therapy 7ith %?, '$or folic acid

A#ailale in #ery fe7 analyzers, not standardized

Case 1
38/! N" #$s%"&' a(a$)a*)e
+,-./023,404,205,607689:;20<=>?@A
Col' agglutinin 'isease
,A$!, H A, !&V B@, !&H ),, !&H& )5.A, RD- ?@.+, Plt
?5>
:lags0 %lasts, .2, n9rc, rc fragments, giant
platelets
Case $
&onclusion0
&e)ere :emol+sis 'ollo9in( Prima<uine
in(estion in 71P! de'icienc+
5* nrc9s$?** -%&
Spherocytes S
2iant platelets
Case 4 : 44*M2 T:rom-oc+topenia = 1 mnt:s2 no
-leedin(0 ll ot:er parameters W8L2 > ITP
Fla(s: n?r-c2 micro r-c* r-c
'ra(ments
Action0
&hange
anticoagulant to
Sodium &itrate
Platelet count- ,@)
&onclusion
=D3A dependant pseudothromocytopenia
4=DP6
=DP
=D3A dependant pseudothromocytopenia
4=DP60
1
Hypothesis- antigen-inding site in the
2P..$...a comple8 , normally
hidden$cryptic, is modified y or e8posed
only in presence of =D3A
1
.n-#itro phenomena
1
Associated 7ith autoimmune$ neoplastic
pathology, ut also seen in healthy
indi#iduals
1
Anormal plt from &!PD, more prone to
clumping y =D3A
1
Alternate anticoagulantsT ?*R trisodium
citrate$ A&D
Case B: ?5$!, :e#er
&onclusion0
Plasmodium falciparum , P. ?5R
3hromocytopenia
!alaria discriminant factorK +.)
3HA/U VE;
rc:ana "a6i'dar2 M0!0
&RL RELI7RE LT!0