ABS Global, Inc. and Genus plc – Ex. 1020, cover 1
`
`

`

`ABS Global, Inc. and Genus plc – Ex. 1020, cover 2
`
`

`

`ABS Global, Inc. and Genus plc – Ex. 1020, cover 3
`
`

`

`RESULTS OBTAINED USING A PROTOTYPE
`
`MlCROFLUlDICS-BASED HEMATOLOGY ANALYZER
`
`Eric Altendorfl', Diane Zebert‘, Mark Hollz, Anthony Vannelli‘, Caicai Wu',
`Thomas Schulte1
`
`1) Micronics, lnc., Redmond. WA 98052, USA
`
`2) Department of Bioengineering, University of Washington, Box 352141,
`
`Seattle, WA 98195, USA
`
`Abstract
`
`Microfluidic laminate-based structures incorporating hydrodynamic focusing and flow
`channels with dimensions much less than 1 mm were fabricated and used to transport and
`analyze blood samples. Optically transparent windows integral to the flow channels were
`used to intercept the sample streams with a tightly focused diode laser probe beam. The size
`and structure of the blood cells passing through the laser beam determined the intensity and
`directional distribution of the scattered light generated. Forward and small angle light
`scattering channels were used to count and differentiate platelets, red blood cells, and various
`populations of white blood cells. All the blood samples used were characterized using a
`commercial hematology analyzer for comparison and validation purposes.
`
`Keywords: cytometry, light scattering, microfluidics, clinical diagnostics
`
`1. Introduction
`
`flow cytometry is an established means of counting and classifying particles
`Optical
`contained within a fluidic sample [1]. One application involves the analysis of a blood
`sample for the purposes of determining the numbers of platelets, red blood cells (RBCs), and
`white blood cells per unit volume. This is a common clinical measurement, and optical
`cytometers have been incorporated into a number of commercial hematology analyzers.
`Recently, microfluidic techniques have been employed for the purposes of developing
`cytometers which require smaller sample and reagent volumes [2-4]. Analytical instruments
`based on these efforts will be smaller and more portable than conventional devices. This
`paper reports results obtained with a microcytometer utilizing laminate-based microfluidic
`structures and diode laser light scattering optics. A small portable hematology analyzer
`incorporating this microcytometer is presently being designed.
`
`Blood cell counting and differentiation by laser light scattering: Blood cells flowing through
`a tightly focused laser beam can be identified by the resultant scattered light. At small angles
`relative to the incident beam, the scattered light is largely a function of blood cell size [5]. At
`larger angles,
`the scattered light
`intensity is also dependent on the degree of cellular
`complexity and internal structure [5]. Terstappen et al. [6] demonstrated that
`the scattered
`light at relatively small scattering angles also contains structural as well as size dependent
`information. In particular, a combination of forward angle light scattering (FALS), which
`deviates from the incident beam direction by less than 3 degrees, and small angle light
`scattering (SALS) which is collected at slightly larger angles (3 to 11 degrees from the
`incident beam direction), can be used to differentiate white blood cells. In addition. either
`FALS or SALS signals can be used to count and differentiate RBCs and platelets.
`
`73
`
`ABS Global, Inc. and Genus plc – Ex. 1020, p. 73
`ABS Global, Inc. and Genus plc — EX. 1020, p. 73
`
`

`

`74
`
`Hydrodynamic focusing: In order to count and classify blood cells in the manner described
`above,
`the cells must be made to pass one at a time through the incident laser beam.
`Traditionally this is done by using hydrodynamic focusing [1], where the blood cell sample
`stream is encircled and combined in appropriate proportions with a sheath fluid stream, and
`both fluids are made to pass through a tapered orifice or flow channel. The hydrodynamic
`forces of the flowing and constricting sheath fluid then cause the sample stream to form a
`narrow thread of flowing cells.
`
`Microfluidic flow cytometry: Microfluidic structures can be used to spatially confine blood
`cells in very narrow and precisely formed channels [3] without the need for a sheath fluid.
`Such microstructures cannot, however, typically accommodate high flow rates due to the
`shear stresses generated. The microcytometer described in this paper spatially confines both
`the sample and sheath streams within a microchannel of appropriate dimensions for the
`assays intended.
`
`2. Experimental
`
`All the experiments were performed in laminate-based planar microstructures manufactured
`by Micronics, Inc. Two representative structures are shown in Fig.
`l and Fig. 2. The device
`shown in Fig.
`l was comprised of two thin glass windows separated by a 100 micron thick
`adhesive layer into which flow channels were cut. This device produced a focusing of the
`sample stream in one plane only, and hence will be referred to as the 2-D flow cell. The
`cross-sectional dimensions of the channel containing the focused cell stream was 100
`microns by 1mm. The structure shown in Fig. 2 was formed from Mylar and Mylar-laminate
`sheets (3M, Austin, TX). The channels were cut with a C02 laser system (ULS—25E, Universal
`Laser Systems, Scottsdale, AZ). This device produced a focusing of the sample stream by
`fully encircling the sample stream with a sheath fluid stream, and will be referred to as the 3-
`D flow cell. The cross-sectional dimensions of the channel containing the focused cell stream
`in this device was 100 microns by 500 microns. A custom control station consisting of two
`computer controlled syringe pumps (Kloehn Company, Ltd, Las Vegas. NV) was used to
`provide constant sheath and sample flow rates in the micro-structures. The sample flow rate
`was 95nl/s in the 2-D flow cells and
`440nl/s in the 3-D flow cells. A 9mw, 685nm
`
`wavelength diode laser module (Melles Griot, Boulder, CO), which produced a near circular
`collimated 4mm diameter beam, was used as the light source. The laser beam passed through
`two crossed cylindrical lenses in order to produce a focused elliptical beam with dimensions
`perpendicular to the direction of propagation of 13 microns by 105 microns at the sample
`stream. Two high speed narrow format seaming photodiode detectors (Centro Vision,
`Newbury Park, CA) were used to collect the PALS (1.4 degrees to 2.2 degrees) and SALS
`(2.2 degrees to 8.4 degrees) signals. An obscuration bar was used to block the direct laser
`beam from impinging on the detectors, and three lenses and a custom mirror with a central
`aperture (hole) were used to direct the scattered light to the detectors. Electronics signals
`were collected with an AT-MIO-l6E-l data acquisition board (National Instruments, Austin,
`TX) when using the 2-D flow cell, and with a with a custom high speed data acquisition
`system when using the 3-D flow cell.
`
`All blood samples (human) were collected with vacutainer (Becton Dickinson, Franklin
`Lakes. NJ) tubes containing the anticoagulant EDTA, prior to preprocessing and dilution. For
`the RBC and platelet assays, dilution of the samples with phosphate buffered saline was
`carried out by external manual mixing prior to introduction to the flow system. For the white
`blood cell assays, external mixing of the blood sample with a commercial soft lysing reagent
`(Snack-Sheath, Streck Laboratories. Omaha, NE) was carried out prior to introduction to the
`instrument. Times between mixing and analysis in both cases were kept to a minimum to
`
`ABS Global, Inc. and Genus plc – Ex. 1020, p. 74
`ABS Global, Inc. and Genus plc — EX. 1020, p. 74
`
`

`

`avoid excess lysing of white cells or osmotic distortion of the RBCs and platelets. Aliquots of
`all the original samples, as well as aliquots of the mixed and diluted samples where possible,
`were analyzed with a commercial hematology analyzer
`(Cell-Dyn 3500R, Abbott
`Laboratories, IL) for comparison and control purposes.
`
`75
`
`
`m1
`‘(—""'"'—'—\,"‘.'
`'»
`;: ¥ (Ti-w
`:39 %l_\
`j»;
`.u V l:
`1 WL “1"].
`i
`PS) i
`f
`
`Fig.1
`
`:3
`
`10mm
`
`3. Results and discussion
`
`Fig. 3 displays results obtained using a 1:400 prediluted sample of whole blood, and a 2-D
`flow cell similar to that shown in Fig. 1. A histogram of the SALS light scattering pulse
`amplitudes is shown indicating a bimodal distribution corresponding to platelets and RBCs.
`Integration of the area under each of the peaks was used to determine the relative percentages
`of RBCs and platelets in the sample. These results along with data obtained from three
`additional samples are plotted in Fig. 4 against RBC and platelet percentages obtained using
`the Cell-Dyn 3500R, Fig. 4 indicates a good correlation between the two methods.
`
`
`
`a” h
`
`3°“ ’
`
`Platelet
`
`Rees
`
`W 20“ -—
`
`E
`
`ton —
`
`“i
`
`1 HL.
`U
`50
`
`.4
`
`.J
`100
`
`A
`150
`
`in
`g
`2 "
`gQ)CL
`on
`E)O
`Q a0
`gg.
`§0
`i
`
`20
`
`n
`
`
`
`1”
`
`I RBC counts
`o Pmm count:
`-—L|M' "suntan
`
`ll
`
`20
`
`.0
`
`W
`
`.0
`
`no
`
`SALS lntendty
`
`Fig. 3
`
`Cell-dyn cell percentages
`
`Fig. 4
`
`Fig. 5 displays results obtained using a whole blood sample diluted 1:50 in the commercial
`sofi lyse reagent, and a 3-D flow cell similar to the one shown in Fig. 2. A 2-D histogram of
`the FALS and SALS data is shown in Fig. 5 and an analysis of an aliquot of the same sample
`by the Cell-Dyn 3500K in shown in Fig. 6. The 3 dominant clusters in each plot correspond
`to lymphocytes (L), monocytes (M), and granulocytes (G). The microcytometer, and
`
`ABS Global, Inc. and Genus plc – Ex. 1020, p. 75
`ABS Global, Inc. and Genus plc — EX. 1020, p. 75
`
`

`

`ll1I1 I
`
`76
`
`
`
`._ i
`(an)9......4___HA....‘l
`
`FALSIntensity
`
`...
`
`U
`
`7' S-\lS intensity (au)
`
`255
`
`CUHPLEX 1T?
`
`Fig. 5
`
`Fig. 6
`
`commercial analyzer produce similar degrees of cluster resolution. The relative cell counts
`(to the total white cell count) obtained with the microcytometer are 27.0% (L), 9.31% (M),
`and 63.7% (G), which are also in good agreement with the Cell-Dyn percentages of 26.9%
`(L), 9.68% (M), and 63.4% (G).
`
`4. Conclusions
`
`The microcytometer described in this paper has demonstrated the ability of counting and
`classifying platelets, RBCs, and various white cell populations by means of laminate-based
`microfluidic flow channels, and light scattering optics. Additional light scattering and data
`analysis channels will be used to extend the capabilities of the microcytometer toward a
`complete blood cell assay including a five-part white cell differential. This microcytometer
`will then be incorporated into a prototype hematology analyzer for field testing.
`
`References
`
`1.
`
`2.
`
`3.
`
`4.
`
`5.
`
`6.
`
`HM. Shapiro, Practical flow cytometry, Third Edition, Wiley-Liss publishers, New
`York, 1995.
`
`E. Altendorf, D. Zebert, M. H011, and P. Yager, Differential blood cell counts
`obtained using a microchannel based flow cytometer, Sensors and Actuators, 1 (1997)
`531-534.
`
`D. Sobek, S.D. Senturia, and M.L. Gray, Microfabricated fused silica flow chambers
`for flow cytometry, Solid-State Sensor and Actuator Workshop, Hilton Head Island,
`South Carolina (1994).
`
`R. Miyake, H. Ohki, I. Yamazaki, and R. Yabe, A development of micro sheath flow
`chamber, Proceedings of the IEEE micro electro mechanical systems workshop, Nara,
`Japan, (1991) 265-270.
`
`M. Kerker, Elastic and inelastic light scattering in flow cytometry, Cytometry, 4
`(1983) 1-10.
`
`L.W.M.M Terstappen, 3.6. de Grooth, K. Visscher, F.A. van Kouterik, and J. Greve,
`Four parameter white
`cell differential
`counting based on
`light
`scattering
`measurements, Cytometry, 9 (1988) 39-43.
`
`ABS Global, Inc. and Genus plc – Ex. 1020, p. 76
`ABS Global, Inc. and Genus plc — EX. 1020, p. 76
`
`