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1Automated blood cell count JP DEFOUR 18/11/17 BHS
Automated blood cell
count
Jean-Philippe DefourClinical Biologist
BHS educational coursesHof ter muschen18 /11/17
2Automated blood cell count JP DEFOUR 18/11/17 BHS
Rosalind Franklin
Tower
inaugurated in
2005
Av. Emmanuel
Mounierlaan 401200 Brussels
Rosalind Franklin Tower : Corelab level -3N
Hof ter
musschen
XVII century
Natura 2000
Av. Emmanuel
Mounierlaan 21200 Brussels
3Automated blood cell count JP DEFOUR 18/11/17 BHS
XN9000 analyser track with 3 analytical modules.
(max: 300 samples /h)
2X SP10 (automated slide maker and stainer)
240 slides made and stained /hour
2X DI60 integrated slide processing system
60 slides read /hour.
Tube sorter for other test
flow cytometry, immunosupressors , buffy tubes
Tosoh G8 (HbA1c) coupled to the track
Full automation
The widespread use of haematology analysers
has led to a major improvement of cellular
haematology
4Automated blood cell count JP DEFOUR 18/11/17 BHS
Turn around time is the key
N=40 tubes from Hematology daily hospital
pre and post analytical is still critical
Nurse 00:04
Sending 00:04
Lab Reception00:12
Analyser 00:10
Informatics, 00:15
Quick and accurate results are the rule
5Automated blood cell count JP DEFOUR 18/11/17 BHS
CELL DYN ®(Abbott)
Analysers on the market
XN® class hematologysystem (Sysmex )
ADVIA 2120 ® (Siemens)
UniCel ® DxH 800Beckman Coulter
1 2 3 4
COBAS M511®(Roche)
slide printing method
combines a digital
morphology analyzer,
cell counter and
classifier
5
For analysers, blood cells correspond to particles that
differ according to various physical parameters
6Automated blood cell count JP DEFOUR 18/11/17 BHS
cobas® m 511 integrated hematology analyzer
Step 2 : printing a monolayer on a slide
7Automated blood cell count JP DEFOUR 18/11/17 BHS
Anticoagulant is ethylenediamine tetra-acetic acid (EDTA)
Preanalytical condition and anticoagulation are important for hematology!
BUT Spurious thrombocytopenia occurs in several
circumstances related to the presence of EDTA
7
B. Bain Blood cells a practical guide 4th Edition
EDTA K2/K31,8mg /ml de sang
+ good morphology
- • reduced cell volume
• platelet activation
• leucoagglutination
• irreversible • auto-antibody
against platelet
Blood film from non anticoagulated venous
blood.
8Automated blood cell count JP DEFOUR 18/11/17 BHS
EDTA pseudothrombopenia EPT
• 0,1-2,0% of hospitalized patients
• 0,07-0,2% of general population
• αIIb/βIIIa complex seems important ( deficient glanzmann thrombosasthenia cannot agregate by the plasma of EPT))
• Sometime multiple…
Thrombocytopenia discovered in a patient may induce
several procedures including unnecessary bone
marrow aspiration or/and PLT transfusion
9Automated blood cell count JP DEFOUR 18/11/17 BHS
Analysers are species specific.
Bird blood Those chicken RBC and PLT are counted like lymphocytes
10Automated blood cell count JP DEFOUR 18/11/17 BHS
International Council for Standardization in Haematology (ICSH)REFERENCE TECHNIQUES
Used by
manufacturer exclusively manual techniques or
semi-automated techniques
fastidious and time consuming
Imprecise ( hemocytometer)
11Automated blood cell count JP DEFOUR 18/11/17 BHS
Techniques from the first half of the 20th century are not always reference techniques
Blood cell counts(red cells, white cells,
platelets): appropriately diluted blood samples and a ruled counting chamber
(hemocytometer)ICSH reference method is single channel aperture impedance method at
serial dilution except for PLT ( flow cytometry)
Hemoglobin concentration:
colorimetrically by the cyanomethemoglobin
method. ICSH reference method Absorbance at 540nm
The heamatocrit
(packed cell volume): 5 minutes ( 3 more if PV patient) high speed
centrifugation ( 10.000-15.000g) of a column of blood in sealed microcapillary
tubes (75mm long with an internal diameter of 1,2mm leaving 15mm
unfilled )ICSH reference method for the PCV is
whole blood hb/ Packed red cell Hb
Reticulocyte counting:
based on supravital staining of
cytoplasmic ribosomal RNA
CLSI RET count is based on new
methylene blue.
The white blood cell differential:
by examining and enumerating by class (eg,
granulocytes, lymphocytes, monocytes) 100 to 200
individual white blood cells on a suitably stained blood
smear.
1956
PLT reference technique is quite recent flow cytometry
12Automated blood cell count JP DEFOUR 18/11/17 BHS
Past and present
13Automated blood cell count JP DEFOUR 18/11/17 BHS
Brief history
13
1956 : Electronic impedance counter Coulter
1970’s: Light scatter technique (e.g., Ortho ELT-8)
1980’s: Cytochemical counter (Technicon H6000)
1990’s : VCS technology of Coulter STKS)
Ohm's law : V=R.I
DC current (I) is constant
V
(tension)
Time
electrolytic solution
Wallace and Joseph Coulter, 1948
14Automated blood cell count JP DEFOUR 18/11/17 BHS
the first counter 1954
15Automated blood cell count JP DEFOUR 18/11/17 BHS
Blood counter evolution
4 parameters:RBC , WBC , Hb , HT
+ 3 calculated MCV MCH MCHC
Coulter Counter S ™
1969
16Automated blood cell count JP DEFOUR 18/11/17 BHS
log normal distribution with return to base line!
PLT volume ranges from 1-20fl
but the upper threshold that discriminates PLT
from RBC may either be at 36 fl
17Automated blood cell count JP DEFOUR 18/11/17 BHS
PLT impedance channel interferences ( e.g; Sysmex XN10)
Microcytes <36-40 fLPlatelet clumps (various size)SchizocytesDyseythropoiesisgiant platelet
Spurious platelet count
18Automated blood cell count JP DEFOUR 18/11/17 BHS
pseudo thrombopenia
plt clumps
giant platelet ( bernard soulier)
sattelitismsattelitism ( CLL case)
When PLT satellitism occurs the PLT count is moderately
reduced (from 50 to 100 · 109/l), leading to
pseudothrombocytopenia in some but not in all cases
19Automated blood cell count JP DEFOUR 18/11/17 BHS
Cytoplasmic fragments of nucleated cells can interfere
Beside RBC fragments or schistocytes, part of the
cytoplasm of abnormal cells was reported as leading
to the elevation of PLT counts, including leukaemic
blasts, monoblasts, or lymphoblasts
NB: Candida, bacteria, plasmodium can do the same
20Automated blood cell count JP DEFOUR 18/11/17 BHS
High WBC count
CLL >200.000 /ul
RBC impedance channel interferences ( e.g; Sysmex XN10)
RBC abn scattergramdimorphic cell populationRBC agglutination
Iron deficiency treated
RBC agglutination
Macrocytes
Spurious erythrocyte countGiant PLTs also increase RBC count
21Automated blood cell count JP DEFOUR 18/11/17 BHS
First half of the 20th century
Calculated indices
MCV (fL) = ____________________HCT (%) x 10
RBC (millions/µL)
MCH (pg/RBC) = ____________________HGB (g/dL) x 10
RBC (millions/µL)
MCHC (g/dL) = ____________________HGB (g/dL) x 100
HCT (%)
Maxwell Wintrobe
(1932)
90
30
33
Today , development of new indices
or parameters outside the classical red blood cell
(RBC) indices, such as red cell distribution width
(RDW), mean platelet volume (MPV), percentage of
hypochromic or macrocytic RBC have led to many studies
22Automated blood cell count JP DEFOUR 18/11/17 BHS
Fals
ely
ele
va
ted
MC
VReticulocytosis
Leucocytosis (if same channel)
cold agglutinines
Myeloma (with rouleaux)
Hyperglycemia (diabetis , perfusion)
Old sample ( >3 day)
2nd sample control
1st sample taken near a glucose
perfusion
induces an increase of
MCV and a reduction of
MCHC
Annales de Biologie Clinique. Volume 70, Numéro 2,
155-68, Mars-Avril 2012
23Automated blood cell count JP DEFOUR 18/11/17 BHS
MCV Delta check
23
| Current – Previous result |
Average
Change in MCV indicates
Transfusion
Sample mishandling
Sample mix-up
> 5%
Example
(93 – 87) / 93 = 7%
Wrong patient
Is it a duck or
a rabbit?
24Automated blood cell count JP DEFOUR 18/11/17 BHS
MC
HC
>3
6g
/dl
(hyp
erch
rom
ia?)
Lipemia/ turbidity
Hemolysis
cold agglutinines
RBC disorders ( spherocytosis, …)
Often falsely modified: Used for
QC purpose (X bar M)
25Automated blood cell count JP DEFOUR 18/11/17 BHS
Brief history
1956 : Electronic impedance counter Coulter
1970’s: Light scatter technique (e.g., Ortho ELT-8)
1980’s: • Cytochemical counter (Technicon
H6000) • Three part differential
1990’s : VCS technology of Coulter STKS)
26Automated blood cell count JP DEFOUR 18/11/17 BHS
Brief history
1956 : Electronic impedance counter Coulter
1970’s: Light scatter technique (e.g., Ortho ELT-8)
1980’s:
• Cytochemical counter (Technicon H6000 Bayer , now Siemens)
• Five part differential
1990’s : • VCS technology of Coulter STKS)
Absorbance ( peroxydase activity)
FS
C (
siz
e)
In the peroxidase channel forward light scatter, largely
determined by cell volume, is plotted
against light absorbance
27Automated blood cell count JP DEFOUR 18/11/17 BHS
ADVIA 2120 ® Siemens
Some
technologies are
still used in
nowadays
instruments
28Automated blood cell count JP DEFOUR 18/11/17 BHS
Brief history
1956 : Electronic impedance counter Coulter
1970’s: Light scatter technique (e.g., Ortho ELT-8)
1980’s: • Cytochemical counter (Technicon
H6000 Bayer , now Siemens) • Five part differential
1990’s : • VCS technology of Coulter STKS)
DC current (volume)
Conductivity (content of
the cell)
( AC high frequency)
Multiangle scatters
29Automated blood cell count JP DEFOUR 18/11/17 BHS
Present Example with Sysmex XN10, Kobe Japan leader in Belgium.
30Automated blood cell count JP DEFOUR 18/11/17 BHS
Linearity limit on the whole range of hematological malignancies
30
31Automated blood cell count JP DEFOUR 18/11/17 BHS
Low Volume
31
cyanide free : Hemoglobin SLS method
SLS method (without KCN)
sulfolyzer reagent that lyse RBC
32
Hb free within plasma is measured
together with that from the RBC, but
its amount ranges from 10 to 40 mg/l
in normal conditions and does not
affect total Hb measurement
• Haemoglobin: increase
• Lipids
• Immunoglobulins (and cryglobulins)
• In vitro haemolysis
• oxyhaemoglobin (high amount)
• Bilirubin (>250–300 mg/l)
• Haemoglobin: spurious decrease
• Coagulation within the sample
• Overfilling vaccum tube
• Veinipuncture near a drip
• Sulfhaemoglobin
Impedance measure but focusing with a fluid ( diluent )
RBC, PLT
hydrodynamic focusing + impedance measure
33
Hematocrit
34
Peak heighVT
VT
V
Ph = k x VERY
VERY = Ph/k
Ph = Impulshoogte
k = Constante
VERY = Erytrocytenvolume
HKT (%) = V / VT x 100
V = Σ VERY = Σ Ph/k
HKT (%) = Σ Ph / (VT k) x 100time
35Automated blood cell count JP DEFOUR 18/11/17 BHS
Laser flow cytometry
35
36Automated blood cell count JP DEFOUR 18/11/17 BHS
Cytométrie de flux par fluorescence
High DNA or RNA content equal high fluorescence. Immature cells? , producing cells?
one unique reagent!
36
Lymphocyte
PromyelocyteMyelocyteMetamyelocyte
Monocyte
Band
Neutrophile Eosinophile Basophile
BlastNRBC
37Automated blood cell count JP DEFOUR 18/11/17 BHS
Complete blood count ( e.g. sysmex XN10)
37
Lyse
(lysercell reagent )
Stain
( fluorocell reagent)
38Automated blood cell count JP DEFOUR 18/11/17 BHS
NRBCs are now always counted ( no interference with WBC) ( e.g. sysmex XN10)
Mo
Ly
gr
Quality and control of data increased dramatically in
many ways, including various internal flagging , graphic presentation of
particle analysis for identification and enumeration of
specific blood components like NRBC.
FLAG: NRBC PRESENT
NRBC are specifically
identified and may be
enumerated using
fluorescence technology
3939© 2008 Universitair Ziekenhuis Gent
Spurious leukocyte counts
Lipids
(Parental nutrition)
Lysis resistant RBC
(HbC, HbS)Nucleated red
blood cells
(Neonates, Path.
circumstances)
Normal
V STOVE BHS XE2100 ( old generation)
40Automated blood cell count JP DEFOUR 18/11/17 BHS
Less spurious leukocyte counts
Lysis resistant
erythrocytes
LipidsNRBC
each large sized particle (greater than the size of a PLT) that is
not destroyed by haemolytic agents can be identified as a WBC
in case of spurious measurements, the degree by which the
count is affected varies with generation or models
41Automated blood cell count JP DEFOUR 18/11/17 BHS
Mentions déviantes
Scattergramme WBC Abn
Leucocytopénie*
Leucocytose*
Présence de NRBC
Mentions suspectes
PLT-clumps ?
WBC flag from WNR channel sysmex
XN10
41
Such WBC scattergrams are also pivotal
for the generation of flags or alarms
42Automated blood cell count JP DEFOUR 18/11/17 BHS
Exemples diagnostiques CBC + NRBC
Diagnostics positifs
Konv.- eenheden:
HGB: 9.8 g/dl
MCH: 30.1 pg
MCHC: 32.1 g/dl
NRBC aanwezig
Basophils not well
differentiated
43Automated blood cell count JP DEFOUR 18/11/17 BHS
6 part DIFF ( including Igs) ( e.g. sysmex XN10)
43
Granuleux immatures
44Automated blood cell count JP DEFOUR 18/11/17 BHS
Flagging system based on abnormal cell population
Left shift
atyical Lymph?
Lympho Blasts/ Abn ?
PLT-clumps ?
45Automated blood cell count JP DEFOUR 18/11/17 BHS
AML
46Automated blood cell count JP DEFOUR 18/11/17 BHS
Reactive lymphocytes.
46
CD3+ / CD8+ / CD38+ / HLA-DR+
EBV / CMV
47Automated blood cell count JP DEFOUR 18/11/17 BHS
CLL
47
48Automated blood cell count JP DEFOUR 18/11/17 BHS
reticulocyte
48
RET HE=
reticulocyte
’s Hb
• Spurious reticulocytes count:
• Inaccurate gating of RBCs: giant PLTs, PLT clumps,
abnormal WBCs, abnormal number of WBCs, WBC
fragments, nucleated red blood cells
• Intraerythrocytic particles: Howell-Jolly bodies,
Pappenheimer bodies, basophilic stippling, Heinz bodies,
sickle cells, spherocytes, Haemoglobin H inclusions,
plasmodium, babesia
• Others: cold agglutinin disease, autofluorescence of RBCs
(drugs, porphyria), paraproteins, haemolysis, diagnostic
intravenous fluorescent dyes
49Automated blood cell count JP DEFOUR 18/11/17 BHS
less interference
correlates with CD61 and CD41 (FCM)
IPF (reticulocytes of the platelets)
PLT-F
49
pseudo thrombocytes ( AML
fragments?)
50Automated blood cell count JP DEFOUR 18/11/17 BHS
antibody method
Cytodiff™, Beckman-Coulter
•6 antibody – 5 colors
•9 - 13 sub populations !
51Automated blood cell count JP DEFOUR 18/11/17 BHS
Automated microscopy : Cellavision DM96
52Automated blood cell count JP DEFOUR 18/11/17 BHS
Future
53Automated blood cell count JP DEFOUR 18/11/17 BHS
Microfluidics-based analyzers
based on size, granularity
Lens-free holographic microscopy
captures the interference pattern (hologram) of scattered and transmitted light of a cell directly on a CMOS imager
Relative simple optic
has great potential for miniaturization and integration into a microfluidic blood analysis platform
Full IMEC Leuvenlens-free microscopy platform
54Automated blood cell count JP DEFOUR 18/11/17 BHS
Example: Imec’s lens-free microscopy platform
Vercruysse D, Dusa A et al, Lab Chip 2015 , 15, 1123IMEC, Life Science Technology Dept.Kapeldreef 75, B-3001, Leuven, Belgium
55Automated blood cell count JP DEFOUR 18/11/17 BHS
Proof of concept data showing a 3-part WBC diff generated with this method
Vercruysse D, Dusa A et al, Lab Chip 2015 , 15, 1123IMEC, Life Science Technology Dept.Kapeldreef 75, B-3001, Leuven, Belgium
34.5%
57.9%
7.5%
Ne Ly Mo
RBC-lysed blood was flowed through the microfluidic systemLens-free images were acquired from each cellImage analysis was performed on each individual cell (feature selection by scale space)Aliquot from same blood sample analyzedon Beckman Coulter LH-750 for comparison
56Automated blood cell count JP DEFOUR 18/11/17 BHS
INVESTIGATING MORPHOLOGY OF ACTIVATED GRANULOCYTE BY LENS-FREE MICROSCOPY
Vercruysse D, Dusa A et al, Lab Chip 2015 , 15, 1123IMEC, Life Science Technology Dept.Kapeldreef 75, B-3001, Leuven, Belgium
• A subpopulation of granulocytes purified by CD15+ magnetic microbeads (Miltenyi Biotec MACS) exhibit a some level of activation apparent in morphological differences (increase in size, more irregular shape)
57Automated blood cell count JP DEFOUR 18/11/17 BHS
in Vivo
Tuchin V, Cytometry, 2011
58Automated blood cell count JP DEFOUR 18/11/17 BHS
QUESTIONS EXAMPLES…
About Hb Channel on new sysmex analyser: a. measure hb of reticulocytes
b. used sodium lauryl sulfate
c. measure absorbance of methemoglobine.
About Sulfolyser reagent ( hb):a. Contains KCN
b. Lyse RBC
c. complex Hb by transforming iron in sulfate.
About Hydrodynamic focusing:a. is used for RBC/PLT impedance channel
b. is used for flurorescent chanel.
c. is used for Hb dosage.
About MCHC:a) is calculated
b) is measured
c) is expressed in %
About hemolyzed sample: a. influence Hb dosage
b. influence % immature granulocytes
c. influence MCHC.
59Automated blood cell count JP DEFOUR 18/11/17 BHS
Thank You