Review 12 worldwide customers. worldwide 200 about by This attended session cytometry. luncheon the flow at France, 2012, Nice, ISLH in at of myself by presented was field article the in works pioneering as considered bio-photonics technologies, edge sophisticated cut more including toward designs evolved then and DIFF, part 3 for Signal) Impedance (Cell CIS on based technologies, robust and simple analyzers. using It is emphasized here waythe started HORIBA Medical is a key which differential, feature for of leucocyte evolution the hematology retraces of technologies article This Philippe NERIN for White BloodCell Differential atHORIBA Medical HORIBA MedicalinEurope: Evolution of Technologies Review English Edition No.39September 2012 the raw data by using a classification algorithm. What definitely definitely What algorithm. mm per whole volumes manage (traditionally accurately to capacity it is cytometer flow classification classic the a from a analyzer new this store using differentiates and by data acquire raw the sensors, several process or finally and mechanisms, physical several one from resulting information toward stream low f specific a a add sample, the dilute within cells the carry detection, for or reagent multiple single parameters cell stopper, hermetic a through blood the sample to able device automated fully a is analyzer hematology a Basically, disciplines and technologies such as biology, biochemistry, chemistry, chemistry, biochemistry, biology, processing. data andstatistical as acquisition data electronics, optics, fluidics, such technologies and various disciplines involving system are complex a is analyzer hematology a microscope) Nowadays, photonic a by back early accuracy. acceptable with account into taken and differentiated (examined populations cellular main the cases, most in since, considerable hematology field of the in allowed projections methods optical and electrical the reagents, specific with along year after year Improved detector. scattering optical first the and gate) electronic (the so-called measurement impedance parallel: developed in were techniques two when century, twentieth mid the to back go cells blood of measurements automatic in attempts first the that out pointing worth is It Introduction apparatuses. Prior to this, it is necessary to point out that leukocyte leukocyte that from originating out cell, stem the cell, single a from point arises blood to circulating in cell Any hematopoiesis. necessary to related is strongly are it counting and this, differentiation to Prior apparatuses. of generation last the toward products ABX of HORIBA beginning technologies the of from evolution the out pointing differential, leukocyte on focus will article this measurement, leukocyte and cell blood red , provide analyzers ABX HORIBA all Although level of automation. high its to added 3 or per µl) per or [1]

Technical Reports Review .

The bone marrow is one of the most complex tissues in the body. Distributed HORIBA Medical in Europe: Evolution of Technologies as red marrow all over the skeleton, but mainly situated in the pelvis area, the sternum and the vertebrae, an adult weighing 70 kg has roughly 1.5 kg for Differential at HORIBA Medical hematopoietic tissue. This comes in addition to 5-6 liters of blood, of which more than 2 kg consist of red blood cells.

Philippe NERIN The main process for blood production is: (1) A few stem cells produce a large number of progeny cells. This goes on continuously, and a single stem cell may, in a week or two, have up to 106 descendants. The stem cells also maintain their own numbers by This article retraces the evolution of technologies for leucocyte differential, which is a key feature of hematology additional cell division, as showed in Figure 1. analyzers. It is emphasized here the way HORIBA Medical started using simple and robust technologies, based (2) Immature cells become mature blood cells which are able to perform on CIS (Cell Impedance Signal) for 3 part DIFF, and then evolved toward more sophisticated bio-photonics specialized functions in the body. designs including cut edge technologies, considered as pioneering works in the field of flow cytometry. This (3) A limited number of cell divisions occur in parallel with maturation, with article was presented by myself at ISLH 2012, in Nice, France, at the luncheon session attended by about 200 a final stage where mature cells lose their ability to divide (erythrocytes) worldwide customers. (4) Mature cells are stored for a while in the bone marrow until they enter the bloodstream to perform their specialized functions in blood or tissues (5) The multipotent stem cells have the ability to direct their development into all subclasses of hematopoietic cell types, including erythrocytes, (, and ), mast cells, and , and , as well as the different types of , including B, T, and NK cells.

A few images of such cells are shown on Figure 1. The main objective of leukocyte differentiation is automatic classifying and counting of the lineages according to their morphology, size or more advanced criteria such

8 DIFF 5 DIFF 3 DIFF

Proerythroblast Monoblast Megacaryoblast

Polychromatic Erythroblast Prolymphocyte Granulocytic Megacaryocyte

Myelocyte Eosinophll

Metamyelocyte Metarnyelocyte Eosinophll

Band Cell

Red Blood Cells Thrombocytes Polynuclear Polynuclear Polunuclear MonocyteLyrnphocyte Eoslnophil

Figure 1 Leukocyte subsets according 3 DIFF, 5 DIFF and 8 DIFF definitions.

English Edition No.39 September 2012 13 Review 14 Review English Edition No.39September 2012

HORIBA MedicalinEurope: Evolution of Technologies for White BloodCell Differential atHORIBA Medical resistor R and feedback resistors R1 & R2. All of these resistors are are resistors these of All R2. & R1 resistors shunt feedback a Rs, and R resistor voltage resistor polarization a of constant A composed gate. the network basic a across supplies measurable signals, voltage pulsed into variations current such converts source current of view, the point constant a as long electronic the as From volume, insulating. pulse slightly cell as that considered with be can cell shown biological accordance in varies experimentally be amplitude can It current the increases impedance. therefore, and, current micro-aperture aperture, the electrical the through to passing obstacle an when creates cell, blood A aperture. the through current are electric continuous a producing electrodes Two aperture ofthe side either on placed aspiration. by the through which pulled in is cell, chamber, a blood a inside particle, placed micro-aperture a inside generated to volume. According particle overall the to related Signal) Impedance (Cell CIS the pulse: electronic an detect generate to and designed gate electronic an on relies principle measurement This CIS for 3Part Differential these how and consumption, laboratory. the in have efficiency improved reagent advances and throughput accuracy, of terms evolving in up been taken been have have challenges how out pointing 10 years, technologies last the how during partly illustrates DIFF 8 to DIFF 3 from progress The reviews. cytometry flow or slides useless reducing results thus positive false minimal a with productivity, laboratory increased for reliable results and interpreted easily robust ensuring while samples, providing abnormal flag to methods diagnosis, affordable and accurate more a a for analyses complex more facing ever enable which are systems developing dilemma: instruments diagnostic of vitro in Today, manufacturers Immature Blasts, Basophils, Eosinophils, (Lymphocytes, Erythroblasts) Granulocytes, cells blood of Neutrophils, lineages Monocytes, eight of classification DIFF: 8 flag to used being parameter LIC leucocytes. The abnormal containing samples Granulocytes). differential (Immature IMG specific a and with Lymphocytes) (Immature IML Monocytes), parameter (Immature LIC IMM including the of Cells, count the including Immature Basophils) Large Eosinophils, Neutrophils, Monocytes, (Lymphocytes, DIFF:Extended-5 lineages of of classification leukocytes five (Lymphocytes, leukocytes of lineages Basophils).five Eosinophils, Neutrophils, of Monocytes, classification DIFF: 5 (Lymphocytes, leukocytes of lineages LMG classification. so–called Granulocytes), and Monocytes three of classification DIFF: 3 following definitions: the according organized been has classification leukocyte diagnosis, of automatic area the In DNAcontent. orRNA/ proteins trans-membrane of like expression molecules some the as Figure 2 Figure , an electric field is is field electric an , Technical Reports

Review HORIBA Medical in Europe: Evolution of Technologies for White Blood Cell Differential at HORIBA Medical

VRes Height

v t Width Pulse height & width processor

H = 70 Blood CIS H = 150 + Lysing reagent H = 280

LYM MON GRA CIS: Cell Impedance Signal 31.4% 15.1% 53.5%

Figure 2 Principle of CIS for 3 Part Differential

optimized to simultaneously gain stable operation against temperature changes of the gate resistance Ra and they are set to optimize the electronic signal to noise ratio. The output signal is measured by using a specific electronic circuit designed to determine gate pulse parameters, namely their heights and widths.

It is worth noting that, whereas pulse heights inform about cell volume, widths of the pulses are used to filter the signal, rejecting undesirable pulses corresponding, for example, to simultaneous crossing of the gate by two cells. The MICROS analyzer uses CIS technology. For 3 part DIFF, a specific reagent lyses red blood cells, but it preserves and prepares WBC membrane to react differentially according to cell features:

- When the lyse reacts with cytoplasmic membranes, it allows release of water-soluble cytoplasm and shrinks the cell membrane around the nucleus. - When the lyse reacts with cytoplasmic membranes, it has an intermediate reaction, maintaining its large size in comparison to lymphocytes. - When the lyse reacts with cytoplasmic membranes, it has a limited reaction due to a molecule in their cytoplasmic structure which protects them from the shrinking action of the lyse. This limited reaction makes granulocytes the largest of the sub-populations in cell differentiation.

After the differential lysing action, the machine analyses the height of each pulse as cells pass through the electronic gate. These pulses are then digitized, grouped according to their size (30 fL to 450 fL), and mathematically processed to create the WBC distribution curve, which is also known as the WBC histogram.

The 3 sub-populations of WBCs are charted and classified according to the

English Edition No.39 September 2012 15 Review 16 Review English Edition No.39September 2012

HORIBA MedicalinEurope: Evolution of Technologies for White BloodCell Differential atHORIBA Medical reinforcing optical extinction, even if we have to recognize that the main main the that scattering. is extinction optical to recognize contribution to have we if even stain a as extinction, black optical Chlorazol reinforcing containing reagent lytic specific a with mixed is blood whole method, this In particles. scattering highly to corresponding scattering poorly are extinction which optical high produce eosinophils and neutrophils whereas particles monocytes and lymphocytes to extinction optical Low corresponds algorithm. specific a by identified are populations Optical and (CIS) sub- several where amatrix in displayed and extinction) optical and Signal (volume parameters two by Impedance described are on cells apparatus, Cell this In based Extinction. combine cytometer to flow specific a hydrofocusing use instruments Medical uniform HORIBA with window rectangular a into flow the cell. gate) inside optical projected (the so-called shaper illumination beam a by is source optical tailored non-coherent a Usually, lamp. semiconductor or thermal the on (not source represented light baseline”. The “the called signal Different electrical stationary a producing transmitted, totally is beam light the photoreceptor. a by measured on depicted are situations extinction) optical so-called power (the loss optical an causing beam, light the with it interacts where gate optical an toward sensor CIS the from travel a experiences cell blood Each 3 Figure of gate, electronic the above just placed gate signature optical an through optical travelling and cells volume electronic of measurement simultaneous through accomplished was step new a view, of point technological a From other and herpetiformis dermatitis syndrome. eg hypereosinophilic labeled causes, unknown diseases, typically (not skin rare vasculitis deficiencies other AIDS), antibody some syndrome, cirrhosis, ler’s liver Loeff lymphoma, eg eg tumors, some syndrome, diseases, Churg-Strauss eg vessels), blood of (inflammation lung include: rarer Other causes reactions. drug and diseases skin common to relation in occur also can It infection. parasitic is cause main the worldwide, whereas modern fever, In conditions. of For hay and range asthma as such diseases allergic are wide causes commonest its a countries, themselves. in occurs between eosinophilia discriminated example, are granulocytes if separate to formulated technique efficient be can diagnostic an improved An Granulocytes. and Monocytes Lymphocytes, provides lyse differential LMG The FROM 3to 5Part DIFF population. on reported as histogram a to according printed are LMG. Results as known also is classification This (150 450 to fL) fL Granulocytes 150 (100 to fL) Monocytes fL 100 to fL) (30 fL Lymphocytes following classes: . Figure 2 Figure Figure 2 Figure , specifying the percentage of sub- each percentage the , specifying . When no cell crosses the optical gate, gate, optical the crosses cell no When . Figure ) is a is ) Technical Reports

Review HORIBA Medical in Europe: Evolution of Technologies for White Blood Cell Differential at HORIBA Medical

No cell in the flowcell Baseline

Poorly Scattering Low Optical cell Extinction

Highly High Optical Scattering Extinction cell

Figure 3 Schematic representation of the optical response to a travelling cell through the optical gate to work out the 5 part DIFF.

The Optical Extinction Phenomenon

While a cell crosses the optical gate, the optical detector is designed to provide a sensitive response to light losses including scattering and absorption.

It is worth to analyse, from a physics point of view, the process of optical extinction as a unique feature in HORIBA Medical blood analyzers. Optical extinction is the capability of any particle to remove light from an original beam. This light removal produces power variation in the transmitted light beam. Two independant mechanisms, absorption and scattering, are responsible for optical extinction. Absorption is mostly a non radiative process reinforced by cytochemistry, most often converting light to thermal energy. Whereas scattering is a very complex phenomenon, involving specific features of the particle itself. For blood cells, optical thickness is so

SCATTERING ( = Light deviation)

Incident Light Beam Transmitted Light Beam

ABSORPTION

Figure 4 Optical Extinction Definition

English Edition No.39 September 2012 17 Review 18 Figure 5 Figure Review From «OpticsofBiologicalParticles»NATO ScienceSeriesvol.238 English Edition No.39September 2012 Dried protein Cortical cytoplasm Cytoplasm Melanin Mitochondria, ratliver hamster ovarycells Cytoplasm, Protein Cytoplasm Lipid rat livercells Mitochondria, rat livercells Cytoplasm, Refractive indexes of biological coumponds (left hand side) , a pseudo realistic model of cell (center), and numerical results of scattering scattering of results numerical and (center), cell of model realistic pseudo a , (bottom). size side) nucleus and (top) hand concentration granule (left inner account into taking model cell coumponds asimplified for diagrams biological of indexes Refractive Cell Component

HORIBA MedicalinEurope: Evolution of Technologies for White BloodCell Differential atHORIBA Medical 1.353-1.368 1.358-1.374 IndexR 1.58 1.42 1.37 1.50 1.35 1.48 1.40 1.38 1.7 (Lanni etal.,1985) (Vitkin etal.,1994) (Liu etal.,1996) (Brunsting andMullaney,1974) (Kohl andCope,1994) (Kohl andCope,1994) (Beuthan etal.,1996) (Beuthan etal.,1996) (Beuthan etal.,1996) (Barer andJoseph,1954) (Bereiter -Hanetal.,1979) eference According to to According X: Y: measurement. extinction components measurement, optical two impedance its by identified is being electronically cell and Each optically analyzer. probed the of performance the light for and responsible strength scattering illumination/detection and of absorption balance they since important very are wavelength, aperture numerical optical as parameters, such involving size tradeoffs nucleus Design on not. is dependant scattering is forward organelle whereas scattering this side on hand, 20 other dependant than the not On is more content. angle, scattering scatter forward high at whereas mainly degrees, effect significant produces concentration higher concentration: organelle or granule cell on dependant realistic cell , wherein nucleus, cytoplasm and granule refractive indexes indexes refractive account. into taken havegranule been semi and a for cytoplasm angle nucleus, scatter wherein , cell versus realistic scattering light of calculations computer the in reported as side ofleft species biological others and mitochondria cytoplasms, for teams research several by measured been have indexes refractive These extensive with index. refractive aspecific using having particle each compartments, different random done pseudo a is been cell have biological A calculations. progresses computer of lot a then, Since scattering. 1907. to in Mie comparison Gustave by solved in definitely been has sphere perfect a by negligeable Scattering often is absorption that small flow cell alignment. Background noise may also be detected in this zone if if zone this in detected be also may noise Background adjusted alignment. cell flow improperly or Cells), Blood Red (Nucleated NRBCs aggregates, empty. be Platelet lymphocytes, small (LL)indicates normally zone the in of cells detection Any should (LL) zone lymphocyte the of side left far The volume. small their of because X-axis volume the of part lower the as well Y-axis as optical of the part lower the in positioned normally are cells These nucleus. large and cytoplasm condensed with cells shaped round small very 2 Figure 5 Figure Figure 6 Figure nucleus granulocytes . On the right side , top and bottom graphs represent represent graphs bottom and top , side right the On . , the blue cluster represents Lymphocytes which are are which Lymphocytes represents cluster blue the , cytoplasm [2] These curves show that light scattering is is scattering light that show curves These Technical Reports

Review HORIBA Medical in Europe: Evolution of Technologies for White Blood Cell Differential at HORIBA Medical

Figure 6 LMNE matrix representation according to CIS and optical extinction measurement.

biological interference takes place in a significant way.

As represented in Figure 6, optical extinction (Y-axis) is plotted against volumes (X-axis) to form an image with several “clusters” identified as follows:

The blue cluster represents Lymphocytes which are very small round shaped cells with condensed cytoplasm and large nucleus. These cells are normally positioned in the lower part of the optical Y-axis as well as the lower part of the volume X-axis because of their small volume. The far left side of the lymphocyte zone (LL) should normally be empty. Any detection of cells in the (LL) zone indicates small lymphocytes, Platelet aggregates, NRBCs (Nucleated Red Blood Cells), or improperly adjusted flow cell alignment. Background noise may also be detected in this zone if biological interference takes place in a significant way.

The purple cluster represents Monocytes which are very large irregular shaped cells with large convoluted nuclei. The nucleus contains folds and sometimes vacuoles. The cytoplasm is also large with non-granular intra- cellular material. These cells will not scatter or absorb a large amount of light when passing through the optical gate. They will therefore be positioned in the lower part of the optical Y-axis. Because monocytes are large cells, they will be placed on the right side of the volume X-axis.

The green cluster represents Neutrophils which are larger in size than lymphocytes. Neutrophils contain granular material in their cytoplasm along

English Edition No.39 September 2012 19 Review 20 Figure 7 Figure Review English Edition No.39September 2012 NA) in comparison with optical Extinction (EXT) which provides High Numerical aperture (High NA) filtering therefore the Scattering profile profile Scattering the therefore filtering NA) (High aperture Numerical High provides which polarizations. TM (EXT) and TE both for Theory Mie from derived Extinction optical with comparison in NA) (SSC)Detector of Scattering atSide placed ofangle right Comparison the (LOWlight beam (top aperture a image) lowrelatively numerical producing SSC EXT

HORIBA MedicalinEurope: Evolution of Technologies for White BloodCell Differential atHORIBA Medical VOL VOL

Intensity (a.u.) Intensity (a.u.) Optical and Scattering Side simultaneously measuring cytometer flow customized a using Medical HORIBA at investigated was EXT versus SSC Side Scattering (SSC) versus Optical (EXT) Extinction detection. for basophil combined are Extinction Optical and CIS where technology OCTRA in made was improvement An sample. the of analysis volumetric for reading CIS on based measurement basophil a allows process this basophils, of cytochemistry on Based shaped. remains which cytoplasm except basophils leukocytes shrink and lyseto erythrocytes used is (ABX BASOLYSEreagent specific order a in II) population, rare this To detect matrix. LMNE the in embedded are they cells, rare are Basophils of leukocytes. (Large spectrum matrix LIC the and Lymphocytes) complete they pathologic in blood, identified cells Cells), other are (Atypical Immature ALY parameters, Additional the of side right the across matrix. population this spread will content organelle granule/ increased of nucleus and the Hyper-segmentation image. the of top the at placed are they cells, other than light more scatter they Because cytoplasm. the within nuclei segmented and material granular contain They neutrophils. like somehow are which Eosinophils represents cluster red The Hyper Y-axis. optical the maturity. on higher their cells these to place will content granule according increased and segmentation X-axis the along up and spreading then concentration, granule to their on cloud the depending up Y-axis the of spreading values gate, higher optical the through pass they as scattered be will light more features, cellular these to Due nucleus. segmented a with scatter angle(degree) scatter angle(degree) Mie diagram Mie diagram Light Beam Light Beam OPTICAL EXTINCTION SIDE SCATTERING HIGH NA LOW NA Latex Bead Latex Bead Technical Reports

Review HORIBA Medical in Europe: Evolution of Technologies for White Blood Cell Differential at HORIBA Medical

· Dodecane · Decane · Nonane · Octane · Heptane · Hexane

· Dodecane · Decane · Nonane · Octane · Heptane Transformation · Hexane

EXT – B. ln (VOL + T) – D IDX = A. ln (VOL + T) – C

A,B,C and D are constants

Figure 8 Calibration of bi-parametric representation CIS x EXT to determine a new mapping involving VOL x INDEX. The calibration process is based on emulsions of organic solvents: hexane (marine blue), heptane (red), octane (green), nonane (blue), decane (purple), dodecane (yellow).

Extinction. Figure 7 is an amazing demonstration of side scattering and extinction behaviors for polydisperse polystyrene beads, diameters ranging from 1 to 20 µm.

The horizontal axis represents particle volume and the vertical axis optical response. Side Scattering detector (SSC) produces a very strange behavior with strong oscillations whereas Optical extinction (EXT) has a smooth like response. Explanations of these results come from Mie theory where it is derived that scattering of a perfect sphere is an oscillating function of size parameter or scatter angle. In SSC, these oscillations are weakly filtered by the low numerical aperture of side optics (pink color). On the other hand, optical extinction takes place over a large numerical aperture, causing an

Normal Abnormal 14300 14300 14250 14250 14200 E 14200 14150 14150 IMG-E 14100 14100 E 14050 14050 14000 14000 13950 N 13950 IMG-N 13900 13900 N 13850 13850 13800 13800 M IMM L L 13750 M 13750

Refractive index (650 nm ) 13700 Refractive index (650 nm ) 13700 IML

13650 500 1000 1500 2000 2500 3000 3500 4000 4500 5000 5500 13650 500 1000 1500 2000 2500 3000 3500 4000 4500 5000 5500 Vol (fL) Vol (fL)

Figure 9 Normal (left) and Abnormal samples (right) according to orthogonal representation volume x refractive index. This classification process was compared to microscopic examination of blood smears, and algorithms were optimized to match manual counts as much as possible. More specifically, we defined thresholds to identify immature granulocytes (green color), monoblasts (grey color) and (blue color). These fundamental results were the starting point for flags on Pentra DX analyzer. Accordingly, ten years ago, HORIBA Medical introduced the extended 5 part diff comprising Immature Granulocyte, Monoblast and Lymphoblast counts based on volume and optical extinction measurements. The sum of IML, IMM and IMG is called Large Immature Cells parameter or LIC parameter.

English Edition No.39 September 2012 21 Review 22 Figure 10  Figure Review English Edition No.39September 2012 Example of two abnormal samples from patients: LAL B (left) and CML (right). LIC parameter is derived as the summation of IML, IMM IMM boxes. IML, of summation corresponding in the as falling events derived is IMG and parameter LIC (right). CML and (left) B LAL patients: leukemia from samples abnormal two of Example

HORIBA MedicalinEurope: Evolution of Technologies for White BloodCell Differential atHORIBA Medical IMG LAL B Pictures, in in Pictures, Pentra DX and Nexus: the LIC Parameter average its and volume its by index. refractive identified be can cell each be and can blood displayed abnormal and normal representation, orthogonal new this In to removed indexes. is of refractive volumes and mapping orthogonal an produce dependence volume where one another into index transformed be refractive can and isoline index refractive volume Each dot. colored a as matrix the specific in a represented with sphere perfect a is particle fluidic each picture, this on see can you As (green). top the to (blue) bottom In etc..). 8 using Figure heptane, EXT x (hexane, VOL solvants organic from calibrate made to emulsions applied specific been have considerations These a therefore producing detector. oscillations, side scattering astandard to comparison in response optical smooth of process filtering efficient and specificity of the LIC parameter were investigated versus IG + BLAST BLAST + IG versus investigated were parameter LIC the of specificity and sensitivity study, of this scope the Within blasts. and granulocytes immature for etiologies different containing cells abnormal of to spectra large selected a were provide samples These samples. abnormal 152 including patients of 218 total a upon based was experiment The Hospital. Bordeaux at samples abnormal detect to analyzers of these capability the Weinvestigated recently LIC The counts. manual and purpose. used flag for showing definitively therefore was parameter automatic counts between cytology to correlations compared acceptable (Large was LIC the and parameter Cells) laboratories Immature a European in biologists including by investigated parameter LIC the show havebeen diseases blood other Many IMG. sides, IMM, of IML, count differential hand right the on and left the on respectively displayed , chronic and Acute analyzers. blood IML LIC IMM , each emulsion have a specific refractive index, increasing from the from increasing index, refractive have emulsion aspecific , each Figure 10 Figure , illustrate some Leucograms of Pentra DX and Nexus DX Nexus and of Pentra Leucograms some illustrate , CML LIC IMG IML IMM Technical Reports

Review HORIBA Medical in Europe: Evolution of Technologies for White Blood Cell Differential at HORIBA Medical FLOW CYTOMETRY (IG + BLAST )

PENTRA or NEXUS (LIC)

Sensitivity 0,94 Specificity 0,83 PV- 0,89 PV+ 0,88

Figure 11 IG + BLAST derived by flow cytometry versus LIC derived by PENTRA and NEXUS.

derived from flow cytometry based on CD16, CHTR2, CD45 and CD11b (standard protocol used at Bordeaux Hospital). We reported that Pentra DX and NEXUS analyzers had a sensitivity of 94 % and a specificity of 83% to flag samples, with a negative predictive value of 89% and a positive predictive value of 88%. For routine purpose, these results are highly satisfactory since we reported that the rate of false negative is lower than 10 %. In addition, it is worth pointing out that the absolute count of Immature cells can be derived by mulytiplying the LIC % parameter by the total count of white blood cells; nevertheless we have to recognize that this parameter is mainly designed to detect and flag abnormal cells in the sample. From a biophysics point of view, it is worth noting that despite the fact that measuring principles are far from each other, analysis being based on volume x refractive index measurements, Pentra DX and Nexus instruments deliver highly correlated results in comparison to flow cytometry immunomarking. Figure( 11) FROM 5 to 8 Part DIFF: OCTRA Technology.

To reduce some discrepancies between routine and CD marker analyses, mainly for absolute count of immature cells and its interference with other leucocyte sub-populations, we recently developped a unique optical device, depicted in Figure 12. In addition to previous physical parameters (CIS, SSC and EXT), the cell signature is enriched by fluorescence measurement of RNA / DNA content. Based on this strategy, called OCTRA technology, we were looking for an improved method of detecting and counting immature cells including immature granulocytes, blasts and erythroblasts.

English Edition No.39 September 2012 23 Review 24 Review English Edition No.39September 2012

HORIBA MedicalinEurope: Evolution of Technologies for White BloodCell Differential atHORIBA Medical use of Thiazole Orange (TO), as exemplified in the next section. the next in (TO), exemplified Orange as ofuse Thiazole the through and measured be can level The of activity. nucleic concentration acid nature their on depending acids nucleic of levels different exhibit also leukocytes cells. Mature immature these in amounts larger therefore found in marrow DNA) and bone nucleic Nucleic (RNA are by content. their acids only, denoted in are occurring normally maturation, cell blood of stages Early DNA strands. to /RNA by hybridization other the On blocked is rotation when strongly fluoresces fluorescence. compound molecular this hand, significant no is there motion: mechanical to converted are photons absorbed and bound Carbon-Carbon the about rotate cycles aromatic two the solution, in Free Analyzer. Pentra the in used Black ground state with emission of a new photon at 530 nm. In OCTRA OCTRA In nm. 530 at photon new a of dye Orange emission technology, Thiazol the with the to state returns it nm, ground 488 at photon a of absorption by excited is on molecule relies fluorescence of of byprinciple amolecule, see light re-emission and absorption The resolution. temporal and spatial popular very of are advantages with together selectivity, and detection sensitivity high oftheir because fluorescence on based techniques Analytical processor. electronic by an digitized is blood signature cell each where analysis space fourth a dimensional form measurements Impedance Cell and Scattering Fluorescence, Extinction, Optical (CIS) measurement. Signal Impedance Cell to combined are measurements optical three These cells. of and signals optical scattering fluorescence both induce to used is diode Laser Blue The measurement. extinction optical for used is chamber flow the through passing beam light red The cell. flow the in beams optical two combines Technology OCTRA 12  Figure In OCTRA technology, whole blood is mixed with a specific reagent reagent specific -  a with mixed is following operations: the blood simultaneously performing whole technology, OCTRA In Leukocyte Classification in OCTRA Technology. Removal of erythrocyte by lytic action action by lytic Removal of erythrocyte 650 nm RCLED Schematic representation of the optical principle used in OCTRA Technology. OCTRA in used principle optical the of representation Schematic (488 nm) SIDE SCATTER STOPPER DIODE LASER 488 nm [3] is used and replaces the Chlorazol Chlorazol the replaces and used is + CIS (532 nm) T.O. FLUORESCENCE Figure 13 Figure (650 nm) OPTICAL EXTINCTION . Once the the Once . Technical Reports

Review HORIBA Medical in Europe: Evolution of Technologies for White Blood Cell Differential at HORIBA Medical

- Chemical poration of nucleated cells for fast staining purpose, while maintaining membrane voltage to accelerate penetration of Thiazole Orange (electropositive ion). - Staining of intra cellular nucleic acids by TO, which is an intercalative dye, highly specific to nucleic acids.

Once incubation completed (a few seconds), the cell suspension is pulled through a flow chamber equipped with the electro-optical device depicted in Figure 12.

In OCTRA technology, multiple cells crossing through CIS or EXT gates are detected by pulse width measurements: undesirable events are filtered based on pulse/height ratio information. The four space analysis is used to design algorithms in a self-learning approach, after intensive testing based on a large spectrum of blood samples including normal and abnormal. In addition to the standard matrix CIS x EXT available in the Pentra instrument, OCTRA technology provides a new matrix where ERB, BASOPHILS, IG and HRC cells are identified.

(a)

Absorption Emission (488 nm) (530 nm)

(b)

100.00 90.00 80.00 70.00 60.00 50.00 40.00 30.00 20.00 (c) 10.00 Intensité de Fluorescence (% ) 0.00 500 520 540 560 580 600 620 640 660 680 700 Lambda (nm)

Figure 13 Simplified Jablonsky diagram of Thiazole Orange dye

(a) and associated fluorescence spectra (c) for λex = 488 nm.

English Edition No.39 September 2012 25 Review 26 Review English Edition No.39September 2012

HORIBA MedicalinEurope: Evolution of Technologies for White BloodCell Differential atHORIBA Medical to point out that mature granulocytes produce a TO baseline from which the the which from baseline TO a produce granulocytes mature that out point to (CIS). We volume have their to respect with neutrophils than response EXT much a exhibit a higher produce they Eosinophils size, granule their to Due SSC response. higher complex, more being levels; EXT and RES medium and (violet) Neutrophils produce bigger, they level, TO being but this produce also (pink) eosinophils granulocytes. other like weak remains level TO the hand, SSC other levels. the higher On produce poly-nucleated, they being but of that lymphocytes, color) (blue as size same Basophils the have roughly level orhigh of TO fluorescence. aweak and CIS produce they state, activation their and maturation their on SSC. Depending and EXT for response bigger a produce they cytoplasm, small having and nucleated, mono- Being NRBC. than size in color) larger are (purple Lymphocytes of part nucleuses; small remaining have the which spaces. measurement blue), four level the weak in ofproduce signals (marine Cells erythrocytes, Blood as Red way Nucleated same the in lysed Being SSC response. higher by their denoted themselves are Platelet aggregates no having fluorescence. level of TO poor color), their by separated (grey easily are material, genetic fragments other and aggregates platelet Stromas, xTO. EXT and 14 Figure 14  Figure Standard bi-parametric representation in OCTRA technology involving CIS x EXT and and EXT x CIS xTO images. involving EXT technology OCTRA in representation bi-parametric Standard displays a representation using standard parameters CIS x EXT EXT x CIS parameters standard using representation a displays EXT TO CIS EXT Technical Reports

Review HORIBA Medical in Europe: Evolution of Technologies for White Blood Cell Differential at HORIBA Medical

IG y = 0,887x + 2,1569 r = 0.91 30

25

20

15

10

FLOW CYTOMETRY 5

0 0510 15 20 25 30 OCTRA TECHNOLOGY

Sensitivity 0,95 Specificity 0,8 PV- 0,88 PV+ 0,85

Figure 15 Immature Granulocyte parameter derived from OCTRA technology in comparison with four color cyometry for CD16, CHTR2, CD45 and CD11b.

immature cells are detected.

Immature granulocytes (black) show higher TO level than mature granulocytes, and high EXT. High CIS responses are produced because of their large volumes. Other large leukocytes are HRC (High ARN Content) (sky blue), which produce high CIS and TO responses. These cells have a high TO level due to their high RNA content.

Monocytes (green), being mono-nucleated and having no granules, produce a weak to medium SSC level and a medium to high EXT, TO and CIS levels.

In OCTRA Technology, the HRC box gathers lymphoblast, monoblast and plasma cells.

We investigated the capability of OCTRA technology to detect abnormal samples containing immature granulocytes. The experiment included 196 patients, selected to provide a large spectrum of abnormal cells containing different etiologies for immature granulocytes, blasts and atypical lymphocytes. Within the scope of this study, sensitivity and specificity of the IG parameter and its flag capability, were investigated versus IG derived from flow cytometry based on CD16, CHTR2, CD45 and CD11b (standard protocol used at Bordeaux Hospital). Based on a cut-off of IG (Octra) > 2% and IG (Flow cytometry) > 2%, we reported that OCTRA technology had a sensitivity of 95% and a specificity of 80% to recognize and flag samples containing immature granulocytes, with a negative predictive value of 88%

English Edition No.39 September 2012 27 Review 28 Review English Edition No.39September 2012

HORIBA MedicalinEurope: Evolution of Technologies for White BloodCell Differential atHORIBA Medical laboratory level. laboratory at studied been have ofleukocytes proteins cytoplasmic or membrane Many for Leucocyte Recognition and Counting Biomolecule Detection in reported IG as cells counting and detecting for both standards cytometry flow meet to technique affordable and simple exceptionally an provide leucocytes of expression measurements DNA / RNA that noting worth is it and application, routine for satisfactory highly are results These 85%. of value predictive positive a and Figure 16  Figure despite unbalanced biomolecule expression. biomolecule unbalanced despite measured correctly is PC-antiCRTH2 where experiment LIF wavelength dual Bottom: cells. non-labeled and labeled of separation poor showing TO –RNA/DNA and –CRTH2 PC5 on based experiment LIF wavelength asingle of Top: demonstration [4] Availability of monoclonal antibodies directed against against directed antibodies monoclonal of Availability

Fluorescence intensity of TO Good discrimination Fluorescence intensity of TO Figure 15Figure Bad discrimination Labeled anti-CRTH2cells Non labeledanti-CRTH2cells Fluorescence intensity Fluorescence intensity of PC5-antiCRTH2 of PC5-antiCRTH2 . Technical Reports

Review HORIBA Medical in Europe: Evolution of Technologies for White Blood Cell Differential at HORIBA Medical

Channel 1 Channel 2

) Noise TO AbAbss λ1 Signal TO p ( FluoFluo PPC5C5 AbAbsbs PPC5 absorption (a.u.) FFluo Relative emission and 400 450 500 550 600 650 700 Wavelength (nm)

Channel 1 Channel 2 Signal

) λ2

TO Abs ( λ1 TO Noise p FluoFluo PC5 AbsAbbs PPC5 absorption (a.u.) FFluo Relative emission and 400 450 500 550 600 650 700 Wavelength (nm)

Figure 17 Illustration of spectral overlaps, single (top) and dual (bottom) LIF experiments.

these proteins allows flow cytometric analysis of leukocyte lineages, mainly based on fluorescence immuno-markers.[5] In this context, the standard diagram is replaced by a similar diagram, where cells are fully described by their antigenic expression. A specific cell is described by a set of CD markers, each having a specific expression. Obviously, this concerns the field of standard flow cytometry, except that at HORIBA Medical, we took into account limitations and drawbacks existing in marketed flow cytometers.[6] A first limitation comes from detection of unbalanced fluorescence signals measured in a multi-channel flow cytometer. To illustrate this pitfall, we carried out an experiment where a weak fluorescence (Phycoerythrin Cyanine 5 coupled to an antibody denoted PC5-antibody) is investigated in the presence of a strong fluorescence background (TO from DNA / RNA). In Figure 16, the situation of PC5-antiCRTH2 is reported here whereas several other combinations were investigated.[6]

Figure 16 (top) reports an experiment using a single wavelength operation showing that anti-CRTH2 is weakly detected in presence of TO fluorescence, while Figure 16 (bottom) reports a dual wavelength excitation exhibiting a larger fluorescence for labeled anti-CHRTH2 cells.

At the top of Figure 17, blue and red curves are respectively the absorption spectrum of TO and that of PC5. Dashed lines are respectively the fluorescence spectrum of TO and that of PC5. Using one wavelength illumination, overlap of TO spectrum in channel 2 induces a background optical signal producing quantum noise for PC5 fluorescence measurement. At the bottom of Figure 17, a second wave is added in the flow chamber at 565 nm reinforcing significantly the PC5 fluorescence strength independently

English Edition No.39 September 2012 29 Review 30 Review English Edition No.39September 2012

HORIBA MedicalinEurope: Evolution of Technologies for White BloodCell Differential atHORIBA Medical Figure 18 Figure Looking at Looking noise to overlaps. signal spectral with the measurements color for flow multi ratio cytometry improve to proposed is illumination multi-wavelength in depicted is concept of previous the A generalisation A Flow Paradigm. Cytometry Wavelength Multiplexing: noise to signal 2. forthe channel ratio extent some to improving thus fluorescence, TO the of Figure 18  Figure Medical promoted the development of a specific laser based on non linear fibers. linear crystal inside optics non on based laser specific a of development the promoted HORIBA Medical selection, and operation multi-wavelength on flexibility provide marker To CD amplitude. and Each wavelength defined of wave specific a operation. by adressed being multi-wavelength requires set-up this Thus, channel. wavespecific and a aspecific to associated is antibody CY5.5 excites CY4. wave The the a set-up, as this In wave CD34 purpose. and CD11b differentiation CD45, leucocyte for protocol CRTH2, standard CD16, example, for measurements involving, This expression CD antibody. unbalanced for used be monoclonal can a scheme to general coupled each 5, channel for Cyanine and (CH4) 4 5.5(brown) channel for (red) 5 Cyanine 3, channel for (orange) 3 Cyanine (CH2), 2 channel for blue) (marine for 2 blue) (CH1)Cyanine (AF405 1 , 405 channel Fluor Alexa fluorochormes: following the adress to λ1 λ Supercontinuum . This scheme is a generalisation of our principle for which each each which for principle our of generalisation a is scheme This . CH1 1 excites wave AF405. The Design principle of a multiwavelength and multispectral detection architecture based based architecture source. detection optical supercontinuum on multispectral and multiwavelength a of principle Design λ2 shows the set-up investigated at research level and corresponding corresponding level and research at investigated set-up the shows source Figure 18Figure CH2 Acousto-optic λ3 modulator CH3 λ4 CH4 λ5 CH5 , let’s consider a fifth channel flow cytometer designed designed cytometer flow channel fifth let’sa , consider Detector Excitation wavelength [7, 8] λ 4 excites CY5 wave the and ~ Synthesized opticalsource λ 2 excites CY3. wave The CH1 flow Cell Figure 18 Figure CH2 Detector CH3C λ Grating 5 excites excites 5 H4 . Here, Here, . CH5 λ [6] 3 Technical Reports

Review HORIBA Medical in Europe: Evolution of Technologies for White Blood Cell Differential at HORIBA Medical

Trigger signal Infrared Pulse

Laser

Output Beam Profiles measured at 3 wavelengths Pump LASER ( 488 nm, 530 nm and 632 nm) 1064 nm

LP01 Mode

Figure 19 The supercontinuum optical source principle used at HORIBA Medical

to a compact solid-state flow cytometer with multiplexing-demultiplexing capabilities for CD expression measurements. Somewhere, it stands as a paradigm in the field of flow cytometry since the device is expected to be fully programmable thanks to a wavelength selector acting as an optical synthesizer tailoring both wavelength and wave amplitude for CD marker expression measurement. Preliminary results of this promising technology have been accepted for publication.[9] In Figure 19, the optical source is depicted. It is based on a crystal fiber is composed of a small silica core surrounded by air galeries. Non linear effect takes place when an optical infrared pulse is sent into the tiny optical fiber core. When the pulse matches some technical conditions defined by the fiber itself, non linear optical processes convert the original pulse to a supercontinuum light source comprising all wavelengths, from the visible range to the short infrared. The beam emerging from this optical source looks like a white laser with a high spatial coherence. This rainbow-like image corresponds to dispersion of the beam by an optical grating. Thus, the white laser beam can be filtered at specific wavelengths providing LP01 modes identically to a conventional laser beam.

Conclusions

WBC differential technology covers a wide range of products at HORIBA Medical, depending on needs, habits and laboratory organization. Micros and Pentra analyzers provide flexible, accurate and reliable solutions for routine hematology, with satisfactory correlations with many references such as manual counts or cross correlated instruments based on physical measurements. We have to point out that incoming OCTRA technology will bring higher insight with improved correlations to flow cytometry «gold standards».

In addition to continuous improvement in biophysical measurement principles applied in routine analysis, HORIBA Medical explores the way cellular immunophenotypes can be carried out using optical wavelength multiplexing. This methodology is expected to fulfill requirements for multiple measurement of biomolecules, both improving cell identification and accuracy. To some extent, wavelength multiplexing alleviates software compensations applied in standard flow cytometers, which are the root cause

English Edition No.39 September 2012 31 Review 32 Review English Edition No.39September 2012

HORIBA MedicalinEurope: Evolution of Technologies for White BloodCell Differential atHORIBA Medical [9] [8] [7] [6] [5] [4] [3] [2] [1] Science, Measuring and Research Montpellier. Medical, atHORIBA the Innovation leading is encompassing he 2003, Since Biophotonics. research and Biophysics Applied in years 20 a experience has He patents. 25 about of inventor and publications peer 20 reviewed about of author is PhD) (MSc, NERIN Philippe author: the About aging. healthy and active promoting while disease chronic against fight their in Community European and WHO the for guidelines driving the improve will are which medicine, preventive and technologies care of clinical efficiency and sensitivity innovative and routine an that expecting requiring are We follow-up disease lives. their along all patients monitore chronic to methodology sensitive and of accurate concept the monitoring. of (MRD) part is Disease MRD Residual Minimal or disorders hematology of diagnosis including applications, clinical in measurements inaccurate of References

publication in Cytometry Part A, (2012). A, Part Cytometry in publication for accepted Illumination, Laser Supercontiuum Triggered a on Based Cytometer Flow Nérin, P. Huss, G. Hilaire, D.Cremien,S. Ledroit, S. P. Brunel, V. Couderc, P. Leproux, Rongeat, N. 4371 (2004) Vol. 19, 12 Issue pp.4366- Express, Optics system. pumping multi-wavelength original using fiber optical dispersive C. P.Roy, normally in generation Labonté, L. supercontinuum White-light V.Tombelaine, P. Froehly, Nérin, Février, S. P.Leproux, V.Couderc, Champert, PA. A Engineering Optical for Society International the of SPIE, Proceedings jitter, low timing and length low coherence with source laser supercontinuum Adjustable Col. and P. Nerin Y. Hernandez, Bertrand, A. Andreana, M. pp.14076-14082 (2011) analyzer. hematology in analyses particle for encoding technique Wavelength P. Nérin, V. Couderc, Cremien, D. Gineys, JP. P. Brunel, Rongeat, N. GEIL leukocyte and GTLLF of the by approach analysis M. a systematic on based cytometry document Areference Jouault, flow marrow: H. bone normal in pathways Husson, five-color differentiation B. and Four- Fossat, C. Lacombe, Francis Feuillard, J. Maynadié, in Durrieu, F. Béné, C. Applications M. clinical Arnoulet, C. and Principles p.1221-1229. 2000, vol. 46, Cytometry: Chimestry Clinical Hematology, Flow Wittwer, Carl and Brown M. Thiazole Dye Biopolymers DNA, and Orange fluorescent the between interactions “The Kubista, M. Svanvik, N. Nygren, J. 2007, p. 19-29. Springer, Particles Biological of Optics Videen, G. V. and Maltsev Hoekstra, A. in Dunn K. Andrew Reports Technical HORIBA Technologies, Related and Biology, Optics Cell Blood D. Lefevre, P. Nerin, , English Edition N° 8, 2004, p. 26-37. 2004, 8, N° Edition , English , Vol. 1998, p. 39-51. 46, . 2010, vol. 7714. Optics Express Optics , Vol. 19 Issue 15, 15, Issue 19 Vol. , , Technical Reports

Review HORIBA Medical in Europe: Evolution of Technologies for White Blood Cell Differential at HORIBA Medical

Philippe NERIN Research Director. HORIBA ABX SAS PhD.

English Edition No.39 September 2012 33