SECTION 2 PRINCIPLE OF ANALYSIS Table of Contents 2.1. Flow Cytometry... 2.2 Measurement Principle of Urine Celis 2.2.1 Amount of Urine and Microscopy 22.2 Outlines of Detection Method... 2.2.3 Composition of the Main Unit 2.2.4 Detection Signals. 2.25 Scattered Light intensity 2.3 Dyeing of Urine Particle 2.4 Measurement Flow. 25 Analysis Parameters 2.5.1 Forward Signal Light and Eleciric Resistance Signal... L244 25.2 Wave Signal of Various Cells. 28.3 Soaterram Patem (Plt Area Varios Ca) 25.4 Gate Chart of Each Parameter... 255 Histograms. 2.6 Terminology... UF-100 8MSection 2 Principle of Measurement 2.1 Flow Cytometry Flow Cytomeiry (FCM) means analysis of properties of particle such as cells by radiating laser beam to the particle and measuring the scattered light and fluorescence obtained there. After fluorescent dyeing of Specific substance of the cell, the cell flows out from the nozzle in one fine surrounded by the sheath fluid. When the focused laser beam is iradiated to the individual cell, the scattered light and fluorescence will be radiated. Cells are analyzed in deta by the scattergram or histogram combining fluorescence intensity and ‘scattered light intensity with fight signals as the parameter (see Figure 2-1), “The light scattered forward ofthe incident light axis ofthe laser beam is called “Forward Scattered Light; FSC", and the size of the cell is expressed in proportion to the light intensity. Moreover, the fluorescence ‘excited from the dyed pigment can quantitatively analyze properties of the cell such as the cell surface antigen, the antigen in the cytoplasm, and nuclei (amount of RNA and amount of DNA) from the characteristic ofa fluorescence marked antibody and a fluorescent pigment. tis said that the analysis with a fluorescent microscope needs 100,000 fluorescent molecules or more for each cel. FCM needs only 3000 - 5000 fluorescent molecules for each cel, therefore FCM is an analyzing method with a very high sensitivity. Full Automated Urine Cell Analyzer UF-100 employs flow cytometry and electic resistance detection method as its principle of measurement, and it clusters the urine cell component in ive kinds (RBC, WBC, epithelial cell, casts, and bacteria) and also enables a quantitative expression. Histogram fu Stat | Stores | ana Laser Boam| oOo —— Jpispay | Fluorescence Scattorgram Sheath | shosth Fluid | Fluid Celi Suspension Figure 2-4 Flow Cytometry 2.2 Measurement Principle of Urine Cells Measurement principle of this analyzer isto irradiate laser beam to individual cell lowing through the flow Cell forming sheath fiow after fluorescent dyeing, and classifying each particle of cell by photo-electron, conversion of forward scatter ligt signal and forward fluorescence signal. 2.2.1. Amount of Urine and Microscopy “View No." is the value when removing the eyepiece of microscope and measuring the diameter of the lens (mm) from inverted direction as shown. F-10081 24 APR, 1996,(Gide View) (Front View) u hi Eye Piece 10x Scale Figure 2-2 Measurement Methods of "View No." “Real view" is a diameter which can be observed in the actual microscope sight, and itis expressed as the "View No.(mm)’ divided by the magnification factor of object lens. ‘To obtain ‘view area’, for example, when "View No.” of eyepiece is 14 mm and magnification factor of object lens is 10 (LPF), View Area = x xp eal Viet «314 x [18 x3 P= 1.54 mm? area of one view is 1.54 mm2. ‘To obtain “view area" when “View No.of eyepiece s 14 mm and magnification factor of object les is 40 (HPF), View Area = 7 x, Bes View 2 5.14 x 14x27 = 0.008 mn? area of one view is 1.54 mmé. "View area" for various type of lens is listed below. [Type of eyepiece with 10x ‘Object lens with 10x (LPF) | Object lens with 40x (HPF) View Nos ta ao (0.096 mn? | Ww Ne-t8 256 mine 0169-2 Wai. 20 0.196 mm? View No. 26.5 (super-wide view) [0.345 mm? J Converting these values into undiluted urine of one view, when the sample urine is centrituged and the ‘supernatant is discarded (16 mL of urine is centrifuged and the supematant is discarded, and residue of 0.25 mL. 's considered to be condensed 60 times) and taking 20 uL of residue on the slide glass covered by a cover slip (22 x 22 mm) and when View No. 14 and HPF are used, 20 yb 22mm Figure 2-3. Slide Glass and Cover Slip F-10051 22 APR. 199620 2B x 28 20 22 x 28 Converted native urine = 0.096 x 60 x 0.24 yl. (HPF) Converted native urine = 1.54 x 60 x 182 ul (LPF) ‘To convert View No. to be analyzed, using the amount of the analyzed urine is 7.83 yiL, is converted, the analyzed view No, will be expressed as follows. ‘Analyzed View No. Analyzed View No, 2.2.2 Outlines of Detection Method ‘UF-100 adopts a detector in which optical method and electric resistance method are integrated (see Figure 2-4). Parallel beam of argon laser focuses into the beam spot by the condenser lens. This beam spot is elliptical and its focused to the center ofthe flow cell. Direct beam from the argon laser is stopped and absorbed bya beam stopper. ‘When sample flows through flow cell, scattered light signal and fluorescence signal are generated by the laser beam. Forward scattered ight is collected by collector lans and led to the detector block. In the detector block, stray light (light not for measurement) is removed by pinhole, and the remainder light is divided into scattered light and fluorescence by a dichroic iter. Scattered light is collected by a lens and led to ‘wave processor’ after photo-electron conversion by a photo diode. Fluorescence is filtered by a Color glass filer and led to “wave processor" after photo-electron conversion by a Photomuttiplier tube. AS {or electric resistance detection method, a pair of electrode is provided at both ends of orifice ofthe flow cell and a constant DC current is conducted between these electrodes. When the particle passes through the orice, electric resistance between both electrodes changes and its detected as a voltage signal and sent to the wave processor. Moreover, the conductivity of each sample is measured, and DC current between electrodes is controlled. Photo-mutiplier Tube Ampitior \7 Erectic Resistance Data Figure 2-4 Principle of Detection 2.2.3 Composition of the Main Unit UF-100SiM 23 APR, 19962.2.8 Composition of the Main Unit (1) Optical System. ‘Optical system is composed of argon laser (wave length 488 nm), laser reflection system, flow cell, forward light collector and sensors (see Figure 2-5). Laser is used as the light source of various FCM (flow cytometry) due to its characteristics such as coherent light. Laser beam is bent by dichroic mirrors and is focused by condenser lens and forms a beam spot. This beam spot is focused at the sample of the flow cell and the forward signal light from the sample is collected by a collector lens, The collected forward light is separated by a dichroic fer nto forward scattered light and forward fluorescence and is converted to the electric signal by each detector; photodiode and photomultiplier. Laser Reflection. ‘Beam Spot Forming System Dichroic Miror ater Figure 2-5 Optical System (2) Hydraulic System (Sheath Flow) Dyed sample is aspirated by diaphragm pump into sheath sample line, and a certain amount of the dyed sample is injected by sheath syringe through sample nozzle (jet nozzle) into the flow cell. At this time, pressurized sheath fluid flows in the flow cell and the dyed sample is led into the center of the sheath flow. Dyed sample particle is ‘drawn into the tunnel (aminar sheath flow) of sheath fluid, without mixing each other. Sample particle is always situated at the center of the sheath. Even a large particle is surrounded by the liquid wall and there is no possibilty to clog. Flowing velocty in the flow cell is approx. 5 m/sec and 10,000 particles can be analyzed ina second. ‘The amount of urine used for analysis is approx. 7.83 iL, corresponding 32.6 views of microscopic analysis of HPF (high power field) of 400x, assuming one view corresponded to 0.24 uL. As. result, small number of sample particle can be detected accurately. ‘This method of laser focusing through the flow cell is called "closed method’, and itis widely used for cell ‘analyzer because of stabilty of sample flow axis. Sheath flow method Increases accuracy and reproducibility of particle counting. It also prevents the generation of abnormal pulse and smudge ofthe flow cell because individual particle flows one by one in the center ofthe flow call, (@) Electronic System Forward scattered light is strong enough that light detector with high sensitivity is not needed and photo diode is used to convert light signal into elacri signal. ‘On the other hand, forward fluorescence ie elight, light detector with high eeneitvity, the photomultiplier, ie used. Photomuitiplier absorbs energy of photon on a photo-electric surface (cathode) due to photo-electric effect of metal, and discharges photo-electron. This photo-electron is accelerated by imposed voltage and is amplified by generation of secondary electron. Thus, forward fluorescence is amplified intensively and is UFA00 SIM 24 APR. 1996After being measured and processed in the wave processor, these electric signals are analyzed and stored by ‘the microprocessor. As for the parameters (scattered ight intensity, scattered light pulse width, fluorescence intensity, fluorescence pulse width, and resistance pulse height) of one particle, scattergrams of various combination of parameters are shown by potting in two-dimensional coordinates. ‘Scattered light intensity and fluorescence intensity combined with the number ofthe discriminated particles also display the histograms (only FBC and WBC). Electric Resistance] Fluorescence |] ‘Scattered Light | | | Fe O sniomiar ©) Pt BE cecrses ; by mm v ee ) | Wave Processor Figure 2-6 Electronic System. 2.2.4 Detection Signals (1) impedance Signal Electric resistance is the numerical value to express the dificuity of electric current in one direction. The electric resistance method detects the change of electric resistance to represent the size ofa particle whan the particle passes through an aperture imposed with electric fied. That is, when a particle passes through an aperture Conducted by DC current, resistance/impedance between electrodes changes. Due to a big diference in conductivity (or, resistance) between the particle and the dient, resistance between electrodes increases when a particle passes through an aperture. This phenomenon can be explained by Ohm's Law, Ohm's Law; \V (voltage) =| (current) x R (resistance) ‘When current “I” between electrodes is constant, voltage "V" depends on resistance "R. Thats, when a particle with a large electric resistance passes, voltage "V" increases proportionally. Thus the voltage depends ‘on size (or, volume) of the particle, voltage pulse measurement gives information of size (or, volume) of the partic. Conductivity of the urine sample varies (about 7 - 40 mS/cm(mili Siemens per centi meter)) and due to dilution factor of 1:4, electric current between electrodes changes regardless of the presence of particle. This affects. UF-100 8M 268 APR. 1996the sensitivity of electric resistance detection and unables to obtain the accurate information of particle volume. So, dilution with URINOPACK diluent (conductivity: approx. 7 mS/em, osmotic pressure: 210 mOsm/kg) confines conductivity of the sample within a certain range, and measuring conductivity before the diluted sample enters the flow cell to correct the detection current to obtain constant sensitivity (see Figure 2-7). Besides this, URINOPACK diluent containg chelating agent (calcium salt is melted by chelating), and due to warming to 35°C during dyeing, crystals in the sample such as amorphous salt (often observed in healthy sample and of lite clinical significance) are removed to prevent to affect the particle measurement, Blctode pemumnal © |) sae fows between eletodes| after compensating for the conductivity ‘ofthe sample. | ‘Conductivity Sensor Electrode} ‘Conductivity of the diluted sample is first measure, end crent oving between GAH Strsietrconrecsnoooome Sheath -G Steins || Flue Died Utne Sample Conduainty Sener Figure 2-7 Flow Cell and Ortice (2) Forward Scattered Light Signal Light is bent its direction by coliding the obstacle such as particle. This phenomenon is called scattering of light. In scattering, electron with small inertia inthe particle resonates with the same wave length as incident light by electromagnetism of light, and the secondary light wave is emitted. That is, the particle is the origin of scattered light and various information of the particle (size, shape, etc.) willbe obtained by observing the scattered light. When particle is spherical, type of scattering depends on the relation between wave length (2) of incident light and particle diameter (4). [© When particle diameter is emalior than wave length of incident light (d = Ae itis called Rayleigh scattering, and scattering in all directions occurs (see Figure 2-8a). In this ‘case, particle is considered to be a point source of light vibrating in an electromagnetic fleld and the scattered light intensity is distributed in symmetry around an axis perpendicular to the incident light. }@ When particle diameter is larger than wave length of incident light (0 > 2): Itis called Mie scattering, and strong forward scattering ooours (see Figure 2-8). When particle Is large, the particle cannot be regarded as an point source of light. Scattering contains more than two waves originated from different points of the particle. In forward direction, waves of the ‘same wave length magnifies each other, whereas in backward direction, waves of different wave length interferes each other. Therefore, stronger scattered light is observed in forward side ot the particle than in backward side. UF-1008M 26 APR, 1996O} Laser Beam| T t Sheath Flow Backward Scattering —, |= Forward Seating a {(b) Mie Scattering (d> 2) 80" VT 100 jo |Sample Flow “(ft Backward Scattering — 1 ——> Forward Soaterng Figure 2-8 Wave Length of incident Light and Scattering Figure 2-9 Flow in Flow Cell by a Particle (k: wave length, d: particle diameter) Various partcies are measured for urine and they show non-symmetty because ofthe same effect as Mie seattering. That is, intensity of scattered light depends on the angle because of interference caused by reflecting and scattering atthe different surface point ofthe particle. And, the angular distribution and intensity of the scattered light depends on the particle size. ‘Size information of the particle is obtained by Using this principle. Moreover, forward scattered light of low angle is strong and itis collected by a lens and size information of {good SIN ratio can be obtained. Laser beam is focused at the flow cell forming an ellipse (see Figure 2-9). ‘When passing this laser beam, the panicle scatters laser beam in various direction. Forward scattered tight is collected by a lens, and wave form is analyzed after photo-electron conversion by a photo diode. (8) Forward Fluorescence Signal Fluorescence is the phenomenon of radiating ight with the absorption of light when light with a certain wave length is iradiated to material. When various fluorescent materials are labeled on a particle to be identified and laser beam is iradiated, the fluorescent material excited by laser beam emis its proper fluorescence. Various information of the particle can be obtained by detecting and analyzing this fluorescence. UF-100 uses two series of fluorescent pigment, phenanthridine and carbocyanine dye. Phenanthridine dye combines spectically to DNA and issues fluorescence of orange color (wave length: 610 nm) with the ‘excitation wave length of 450 - 580 nm (see Figure 2-10). Phenanthridine dye is inserted between bases (of DNA of the cell and it specially dyes DNA. Carbocyanine dye is used as a pigment to dye the ‘cytoplasmic reticulum and it dyes the cell membrane. Carbocyanine dye issues fluorescence of green color (wave length: 505 nim) with the excitation wave length of 420 - 500 nm (see Figure 2-10). Carbocyanine dye has excellent permeabiity and itis absorbed to cytoplasm endospore lipid layer and dyes the membrane. These fluorescent dyes are excellent with short reaction time and small background fluorescence (fluorescence originated by pigment itself). Particle dyed with fluorescent dye issues fluorescence proportional to the concentration of fluorescent ‘material. Fluorescence has generally longer wave length and much weaker intensity than those of incident light. Therefore, in order to detect the fluorescence, stray light (ight not for measurement) is removed by pinhole to reduce the influence of passing light, and green fluorescence is selected by a color glass fier and a dichroic fiter. To detect the slight fluorescence, the photomuitiplier converts the weak light signal to greatly amplified electric signal. Information on the dyeing intensity or dyed length ofthe cell is obtained. by analyzing the fluorescent signal and the cell is discriminated. Exchation wavelength (———) Fluorescence wave length ( Wave Length 300. 400. 500 600 Phenarthisne dye <-————> Carbecyarine dye —_— See Figure 2-10 Excited Wave Length and Fluorescent Wave Length UF-1005M a7 APR. 19962.2.5 Scattered Light Intensity ‘Scattered light intensity is proportional to the particle size i its size is within the size of the beam spot (see Figure 2-11). oan | WORN S eee However, this relation does not apply in larger particles than the beam spot. When width of the beam spot is. ‘expanded to measure EC (size approx. 10 - 160 ym) or CAST (size approx. 20 - 300 jm), scatter intensity of large: size particle and that of small size particle are so different that its dificult to measure together. Therefore, instead of changing the beam spot width, another method of detecting the size is adopted, a method of detecting the time to pass the beam spot (see Figure 2-12). Figure 2-12 Particle Passing the Beam Spot In the UF-100, larger beam spot is used than in the R-2000/R-3000 as shown below because the UF-100 ‘measures larger particles. Light Intensity Time Light intensity 166 um ‘285m _ yt (R-2000/R-3000) (UF-100) Figure 2-13 Comparison of Beam Spots UF-100 51 28 APR, 19962.8 Dyeing of Urine Particle Figure 2-14 Linkage of Fluorescent Dye UF-100 analyzes urine cet! components using two kinds of fluorescent dyes. One is phenanthridine dye which dyes nucleic acid and issues fluorescence of orange colar. This fluorescent dye is inserted and links the adjoining base pair of DNA as shown in Figure 2-14. Fluorescence intensity is proportional to the ‘amount of DNA and material containing much DNA (such as nucleus of cell) issues strong fluorescence. However, this fluorescent dye has poor permeability of the cell membrane resulting poor dyeing intensity for undamaged particle such as live cell. Another fluorescent dye to compensate this is carbocyanine dye which dyes cell membrane. It dyes cell membrane with negative electron charge such as nuclear ‘membrane or mitochondorion and tt issues the fluorescence of green color. By using these two fluorescent dyes, urine cell components are clustered easily UF-100 SM 29 APR. 19962.4 Measurement Flow [a ne on Shae can oer sane | + (enw ceva (saws pee [immer “reo whot owe URINOSEARCH escton Unt (Temperate cnt, Ming, isn) caaahey oo ta faa) eee | ae eee ee i a Ee) | | | Forward fuoresont dts Ea =| E9 eae | ee Sree rane ‘Puowerce erty I ‘Pn Hea Pinna ‘aod | Divay Figure 215 Measurement Flow ‘The sample is diluted into 1:4 and is dyed, 9.0 ui is dispensed by sample syringe in 23 seconds for ‘measurement. In the first two seconds, "H-BACT" measurement in high sensitivity is performed. This is for smaller Bacteria (approx. sum, typical Bacteria: 2-3 um) After intalzing in the following one second, normal ‘measurement is performed for 20 seconds for 7.83 .uL. igh Sensitivity initiliz- ‘Analysie ing Normal Measurement 23 600 Figure 2-16 Measurement Timing UF-100 5M 240 1 see 20sec __| APR. 19962.5 Analysis Parameters 2.5.1 Forward Signal Light and Electric Resistance Signal Height and width, ete. of forward signal light (onward scatter light and fluorescence) and electric resistance pulse signal (Imp) are analyzed to obtain information such as particle size and cross section, dyeing ‘sensitivity, dyed part length, and volume. Thus the characteristics of signal wave of each particle is ‘numerized and the particles are clustered by the classification algorithm. rs “ Piso Signal Resistance puse the a volun via cle reaiance signal ls much bigger compat Wath Fao and vegas a mee Forward Scatter Light \@| °) Forward Seater Light vise ro) f 1 Fluorescence + Pulse Wietn (F¥) Forward Scatter Lig Garena Seater urocen gent ect den intensity a Fite dyes enh Formard Scatter Light represents parle sie, of etc Fw ts computed as follows Fee the cross sectional area ofthe particle, ‘and Fscw the particle length. Facw is computed r= (BW. as follows. (Vv - (cueW. Foon ( -£ts8W_) Figure 2-17 Analysis Parameters UF-100SM at APR. 19962.5.2 Wave Signal of Various Cells “The following signal waves are obtained from forward scatter signal, forward fluorescence and electric. resistance signal. RBC * Horizontal axis of each signal represents time and ‘vertical axis the signal strength. + Puls width from actual Resistance Pulse is bigger Computed we eso Foran Scat ght Sonat rescence Forward Scatter Light Signal Fluorescence Signal Resistance Pulse Signal CAST containing CAST contain no organized stuctues organized sructures Eothetal Cot p Forward Seatior ght Signal Fluorescence sistance Pulse Signal Figure 2-18 Schematic Diagram of Signal Wave of Cell UF-100 SM 242 APR. 19962.5.3 Scattergram Pattern (Plotted Area of Various Cells) Wave Form Processing Figure 2-19 Wave Form Processing and Scattergram F-10091 243 APR, 1996No. 1 Sereen: Area of Each Particle on the scattergram No, 2 Screen: Area of Each Particle on the scattergram Foc, Few Measuring Parameters Analysis Parameters _ Flagging Parameters. * Feo (Forward Scatter Light Intensity): Particle Size REC = Path. CAST (Containing + Fscw (Forward Scatter Light Pulse Width): Particle Length + WBC ‘organized structures) + Fl Fluorescence intensity): Dyeing Characteristics + Epithetiat Col + SRC (Small Round Cel) + Fhw (Fluorescence Pulse Width): Dyeing Part Length CAST (Containingno + XTTAL (Crystal) + Imp (Impedance): Paticle Volume organized structures)» YLC (Yeast Like Fungi) BACT + SPERM (Spermatozoa) Figure 2:20 Explanation of Scattergram (1) RBC (red dot) RBC is distributed in the area shown as RBC of the scattergram (No. 1 and No. 2 screens). RBC in urine is ‘approx. 8 um in cell diameter and has no nucleus. Only the cell membrane is dyed by this analyzer, therefore fluorescence is weak and distribution position of fluorescence intensity is low. IBC in urine ‘shows various shape due to mechanical damage or iliness, resutting various distribution of forward scatter intensity. Especially, when there are many smal size RBC such as glomerulus origin, forward scatter intensity is distributed in low range. In this case, hemolysis or small RBC is often distributed in the lightly shaded area of No. 2 scattergram (FUFscw). And REC size distribution can be confirmed by Fsc histogram. (2) WBC (blue dot) WBC is distributed in the area shown as WBC of the scattergram (No. 1 screen: Fsc/Fi2). WBC in urine has nucleus atthe center and is approx. 10 um. The nucieus is dyed well by this analyzer, therefore ‘luorescence is strong and distribution position of fluorescence intensity is high. WBC in urine also shows various shape as well as REC, resulting wide range of distribution of both forward scatter intensity and fluorescence intensity. Cell volume and density of ive WBC varies lite, resulting strong scatter intensity ‘and weak fluorescence intensity. Whereas cell volume and density of damaged or dead WBC often varies, resulting weak scatter intensity and strong fluorescence intensity. (8) Bacteria (green dot) Bacteria is distributed in the area shown as BACT of the scattergram (No. 1 and No. 2 screens). Dead bacteria is dyed better and shows stronger fluorescence than live bacteria, however scatter light intensity is weak because size of bacteria is small. Especially, detection of spherical bacteria is not possible unless they form cluster. UF-1008M a4 APR. 1996(4) Yeast Like Cell and Spermatozoa (green dot) ‘These are distributed in the area shown by YLC and SPERM of the scattergram (No. 1 screen: Fso/Fi2). Fluorescence intensity of yeast lke fungi is fairly strong showing intensity between RBC and WBC. Sprouting and sporic fungi shows even stronger fluorescence. Spermatozoa is also dyed well and its fluorescence intensity matches that of yeast like fungi. As for the forward scatter light intensity, yeast lke ‘fungi shows similar distribution as IBC and spermatozoa shows lower distribution. Yeast lke fungi can be discriminated from spermatozoa considering that pulse width of scattered light of yeast ike fungi Is smaller than that of spermatozoa. (6) CAST (CAST: green square, Path. CAST: red square) ‘These are distributed in the area shown by CAST and Path. CAST of the scattergram (No. 1 screen: FiwFsow). Impedance signal of electric resistance and forward scatter light are used as parameters to identity the cast and the mucous thread. Impedance represents volume of particle, and forward scatter light represents size of patticle. These information classifies one from the other. CAST and Path. CAST are distinguished by the presence of an organized structure in the cast membrane. CAST (containing no organized structures) contains only a small amount of granule in the substrate with ite or weak fluorescence. On the other hand, Path. CAST (containing organized structures) contains epithelial cell, WBC, and granule, etc in the membrane emitting intensive fluorescence. Classification of CAST and Path. ‘CAST is possible by this information of fluorescence. Path. CAST is displayed by a red square. (6) Epithelial Cell (black dot) Epithelial ces are distributed in the area shown by EC of the scattergram (No. 1 screen: FlwFsow). Large cells such as squamous epithelial cell or transitional epithelial cell are distributed in this area. These epithelial cells show intensive forward scatter light and fluorescence, over the range of No. 1 screen scattergram (Fsc/Fi2). In No. 1 screen scattergram (FiwFscw), epithelial cells are distributed in the area of large pulse width of fluorescence, and smaller pulse width of forward scatter light than that of casts, (Small ound Cell is shown by a black square.) (7) Crystal (yellow dot) Crystalis distributed in the area shown by XTAL of the scattergram (No. 2 soreen: Fsc/Fl). Crystal is not dyed and does not emit fluorescence, and itis distributed where fluorescence intensity is lower than that of RBC. Scatter light intensity is distributed in a wide range because of various size and shape. Calcium oxalate crystal etc. have a tendency to distribute along Fsc axis of the scattergram. And Lurie acd crystal has the tendency to have a distribution similar to that of REC. However, discrimination from FIBC is possible considering that the distribution center of crystal is not steady. Also amorphous salts (phosphate salt, uric sat) which affect the measurement is dissolved and removed by chelating agent in dduent and by incubating (35°C) during dyeing. (8) Others (yellow dot) ‘Small sized epithelia! cells (transitional epithelial cell and its medium - deep layer, renal tubular epithelial ‘cell, et.) are similar to WBC in size and dyeing characteristics. tts appearance is marked with flagging by combining parameters (Forward Scatter Light intensity, Fluorescence Intensity, Forward Scatter Light Signal Pulse Width, Fluorescence Signal Pulse Width, and Impedance). UF-100 SM 24s ‘APR. 19962.5.4 Gate Chart of Each Parameter Gate chart (area where the peak of distribution is enclosed) of each parameter s as follows. Gate of each parameter is fixed by the software. However each parameter wil be scanned untl the value is zero, or ‘minimum level (valley point) with other parameter when overlapped with other parameter. Gate and scanning area are same in "CAST" and SRC". “WBCs" are detected in WBC GATE in second screen (Fiw-Fscw screen) and result is appeared in frst screen (Fsc-Fi2 screen) in blue dots. YLC and Sperm are distinguished by the Fscw signal. Total number of RBC is obtained by the following equation. Total RBC = RBCt + RBC2 + {(number of particle in the area of RBCS excluding RBC! and RBC2) (and) RBCS) (Purpose of the Gate of FIBC2 aims the distinction from yeast lke cell.) vie Peak of the distribution Fsc —————— Path. CAST Fi Fu a ——f | ad cnet Focw Figure 2-21 Gate Chart of Each Parameter UF-100 5M 246 APR. 19962.5.5 Histograms ——= No. of Paticies FSC + Forward Scatter Light intensity Figure 2-22 Histogram (1) RBC Histogram When abnormality of RBC shape appears, average value of forward scatter light intensity (FSC) becomes low. treo) tRecl r Fac Fsc ‘an example of non-glomenuius ‘An example of glomeruius oign origin RBC came 0 FBG carne outs Figure 2-23 Histograms of RBC (2) WBC Histogram WBC which ate bacteria has strong luorescence intensity. WBC which is damaged or is nucleus is naked has weak fluorescence intensity and weak scatter light intensity. twee) Swecl, FSC Fsc ‘An example of WBC that ate ‘An example of denatured ‘YLG (Yeast Like Fungi came out. (degenerated) WEG came out. Figure 2-24 Histograms of WBC F-10051 247 APR, 19982.8 Terminology (1) FsoiFl scattergram. Fsc: Forward Scatter Light intensity Fi Fluorescence Inter (2) Fiw/Fsow scattergram Five Fluorescence Pulse Width Fscw: Forward Scatter Light Pulse Width (8) Analysis Parameters RBC: Red Blood Cell (erythrocyte) ‘wet: White Blood Cell (leukocyte) EC: Epithelial Cell CAST: Cast (total = CAST + Path. CAST) BACT: Bacteria (bacillus) (4) Flagging Parameters Path. CAST: Pathological CAST (Cast with inclusions) SRC: ‘Small round cell Yc: ‘Yeast Liked Cel (Yeast Like Fungi) XTAL: Crystal ‘SPERM: Spermatozoa (6) Erythrocyte Shape information RBC-Info. (RBC-Information) Dysmorphic?: ‘Shape is not similar. Isomorphic?: ‘Shape is almost the same. Mixed?: Mixture (Dysmorphic + Isomorphio) form The glomerulus origin and non-glomerulus origin are presumed by distribution of the Fsc histogram of REC. When erythrocyte of 80% or more exists from the first to 126-th channel of Fsc with small size in Fsc histogram, UF-100 flags “Dysmorphic?" when there is possibilty of the glomerulus origin RBC appears. When erythrocyte of 80% or more exists from the 84-th channel to the 250-th channel, UF-100 flags "isomorphic?" when there is possibilty of non-glomerulus origin RBC appears. Moreover, "Mixed? is flagged when both criteria are 80% or less. Dysmorphie RBC. |somorphic RBC IBC Fc Histogram RBC Fec Histogram eich 250ch wo (um) When 80% or more RBC exists in 1 80% oF exists in the range between Och and 126ch the range between sch and 28h Figure 28 Dysmorphic RBC? Figure 2:29 tsomorphic RBC? “ch(channel) : The numerical index representing the strength or width of detected pulse. Fic range is in 0-255 ch. F-10051 248 ‘APR. 1996(6) Cross-checking OB/Hb: ‘Occult blood/Hemoglobin Leukocyte Esterase Protein Nitrite Related parameters are compared between the UF-100 data and the Dipstick unit data as follows. (B/D + RBG, L.Est + WBC, PRO <> CAST, NIT <> BACT) {As for the setting display of cross-checking, the agreement area is displayed with green color, and the disagreement area is displayed with white as shown in Figure 2-30. If the cross-checked data is disagreed, UF-100 displays "2" mark on the Dipstick data and suggest operator to REVIEW and manual measurement. (OBmH O12 345 678 UF-100 Bc CJ oisagreement Bf Agreement Cross-Checking Figure 2-30 Cross-Checking Setting Screen (7) FVFsow scattergram FL I: Fluorescence Intensity Feew: Forward Scatter Light Pulse Width Fscu Figure 2-31 FEFscw Scattergram UF-100 5M 249 APR. 1996(8) Fsc/Fl scattergram Fsc: Forward Scatter Light Intensity Fe Fluorescence Intensity Fsc Figure 2-82 Fsc-Fi Scattergram (@) FBC Fc histogram: ‘The one of scattered light strength of the erythrocyte that the histogram was displayed. (10) WBC Fsc histogram: The one of scattered light strength of the WBC that the histogram was displayed. wee Fee, ‘250ch - “250ch Figure 233 RBC Fsc histogram and WBC Fic histogram (11) The flag output refers to the distribution condition of the count in scattergranvhistogram and the fixed ‘quantity for each particle count. Total Count: Number of total particles ‘Total of particles of about 2-250 yum which are included in the analyzed diluted sample of 9 uL. Since salts are dissolved, Judgment of normal or abnormal is possible from this data, Path. CAST: Number of CAST particles which contains organized structures. XTAL: Number of crystals SAC: ‘Number of round smal cells SPERM: Number of spermatozoa's Yuc: ‘Number of yeast lke fungi H-BACT Count: Number of bacilus which are counted inthe high sensitivity mode. OTHERS: Number of particles that are not clustered in any of the above categories ‘80% or more of the RIBC's are included in the range between Och and 126ch ‘80% or mote of the RECs are included in the range between 84ch and 250ch ‘Number of BC's that is clustered higher Fsc (RBC’s which distribute in the gate RBC!) Non-Lysed RBC%: Rate of Non-Lysed RBC# to all RBC REC-Mf: BC-Middle Fluorescence intensity REC-Mfsc: RBC-Middle Forward Scattered Light Intensity RBC-FLOWSD: RBC Fluorescence intensity Distribution Width SD ‘The width of the erythrocyte fluorescence distribution in the height of 20% from the lower side to the assumption of the height of the peak of Standard Deviation and the fluorescent strength distribution erythrocyte fluorescent strength width-distribution 100%. (13) WBC Analysis Data WBC-Misc: WBC-Middle Forward Scattered Light intensity F-10097, 220 ‘APR, 19961) Total Count | Path.CAST x TAL ‘SRC SPERM Yuc H-BACT Count OTHERS, (12) Dysmorphic RBC 'somorphie RBC Non-Lysed RBC# Non-Lysed RBC% REC-MFI RBC-MFse RBC-FI-DWSD 0.0 ch) (13) (14) WBC-MFsc Conductivity 0.0 fen] 0.0 [Sen (14) Conductivity Figure 2-34 Analyzed Data ‘Conductivity isan index which shows that itis a flow of electricity atthe conductor (material which leads electricity well easly. Conductivity represents the easiness of electric curent, and Siemens is used as the unit Siemens "S": Conductance between two points when voltage between two points of conductor is 1 volt “V" where 1 ampere “AY of current is conducted. 18 = 1.041 (moh) UF-100 SM 2a APR. 1996