Automated blood cell count

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


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


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


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


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


cobas® m 511 integrated hematology analyzer

Step 2 : printing a monolayer on a slide


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.


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


Analysers are species specific.

Bird blood Those chicken RBC and PLT are counted like lymphocytes


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)


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


Past and present


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


the first counter 1954


Blood counter evolution

4 parameters:RBC , WBC , Hb , HT

+ 3 calculated MCV MCH MCHC

Coulter Counter S ™

1969


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


PLT impedance channel interferences ( e.g; Sysmex XN10)

Microcytes <36-40 fLPlatelet clumps (various size)SchizocytesDyseythropoiesisgiant platelet

Spurious platelet count


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


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


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


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


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


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?


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)


Brief history



1980’s: • Cytochemical counter (Technicon

H6000) • Three part differential

1990’s : VCS technology of Coulter STKS)


Brief history



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


ADVIA 2120 ® Siemens

Some

technologies are

still used in

nowadays

instruments


Brief history



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


Present Example with Sysmex XN10, Kobe Japan leader in Belgium.


Linearity limit on the whole range of hematological malignancies

30


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


Laser flow cytometry

35


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


Complete blood count ( e.g. sysmex XN10)

37

Lyse

(lysercell reagent )

Stain

( fluorocell reagent)


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)


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


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


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


6 part DIFF ( including Igs) ( e.g. sysmex XN10)

43

Granuleux immatures


Flagging system based on abnormal cell population

Left shift

atyical Lymph?

Lympho Blasts/ Abn ?

PLT-clumps ?


AML


Reactive lymphocytes.

46

CD3+ / CD8+ / CD38+ / HLA-DR+

EBV / CMV


CLL

47


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


less interference

correlates with CD61 and CD41 (FCM)

IPF (reticulocytes of the platelets)

PLT-F

49

pseudo thrombocytes ( AML

fragments?)


antibody method

Cytodiff™, Beckman-Coulter

•6 antibody – 5 colors

•9 - 13 sub populations !


Automated microscopy : Cellavision DM96


Future


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


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


Proof of concept data showing a 3-part WBC diff generated with this method


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


INVESTIGATING MORPHOLOGY OF ACTIVATED GRANULOCYTE BY LENS-FREE MICROSCOPY


• 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)


in Vivo

Tuchin V, Cytometry, 2011


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.


Thank You

Automated blood cell count

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